Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences
http://www.biotechniques.com/biotechniques/BiotechniquesJournal/2008/September/Coupled-rolling-circle-amplification-loop-mediated-amplification-for-rapid-detection-of-short-DNA-sequences/biotechniques-45312.html?autnID=591508
http://primerexplorer.jp/elamp4.0.0/index.html
Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue
http://www.biotechniques.com/biotechniques/BiotechniquesJournal/2009/March/Colorimetric-detection-of-loop-mediated-isothermal-amplification-reaction-by-using-hydroxy-naphthol-blue/biotechniques-118324.html?autnID=598661
Tuesday, July 14, 2009
Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences
BioTechniques, Vol. 45, No. 3, September 2008, pp. 275–280
Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences
Jennifer Y. Marciniak 1, Andrew C. Kummel 2, Sadik C. Esener 3, Michael J. Heller 1, 3, Bradley T. Messmer 41, Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
2, Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA
3, Department of Nanoengineering, University of California San Diego, La Jolla, CA, USA
4, Rebecca and John Moores Cancer Center, University of California San Diego, La Jolla, CA, USA
Circularizable oligonucleotide probes can detect short DNA sequences with single-base resolution at the site of ligation and can be amplified by rolling circle amplification (RCA) using strand displacing polymerases. A secondary amplification scheme was developed that uses the loop-mediated amplification reaction concurrent with RCA to achieve rapid signal development from the starting circular molecules. This isothermal reaction was found to be significantly faster than the comparable hyperbranching amplification method and could detect 100 circular copies in less than 1 h.
Coupled rolling circle amplification loop-mediated amplification for rapid detection of short DNA sequences
Jennifer Y. Marciniak 1, Andrew C. Kummel 2, Sadik C. Esener 3, Michael J. Heller 1, 3, Bradley T. Messmer 41, Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
2, Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA
3, Department of Nanoengineering, University of California San Diego, La Jolla, CA, USA
4, Rebecca and John Moores Cancer Center, University of California San Diego, La Jolla, CA, USA
Circularizable oligonucleotide probes can detect short DNA sequences with single-base resolution at the site of ligation and can be amplified by rolling circle amplification (RCA) using strand displacing polymerases. A secondary amplification scheme was developed that uses the loop-mediated amplification reaction concurrent with RCA to achieve rapid signal development from the starting circular molecules. This isothermal reaction was found to be significantly faster than the comparable hyperbranching amplification method and could detect 100 circular copies in less than 1 h.
Cloning improvements
BioTechniques, Vol. 47, No. 1, July 2009, pp. 609–615
Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers Hirokazu Takahashi 1, Kimiko Yamamoto 2, Toshio Ohtani 1, Shigeru Sugiyama 11, NanoBiotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, Ibaraki, Japan
2, Insect Genome Laboratory, National Institute of Agrobiological Sciences, Ibaraki, Japan
The predominant method for DNA cloning is by propagation in biological hosts, but this method has limitations because certain sequences are difficult to clone using any combination of available hosts or vectors. Recently, multiply-primed rolling circle amplification (MPRCA) has been applied to overcome the problems of the DNA cloning via host cells. However, when MPRCA is used to amplify from minute quantities of DNA template, the products are mostly by-product DNA molecules generated by false priming and primer dimer formation. This study demonstrates that MPRCA using random RNA primers—instead of DNA primers—blocked the synthesis of by-products and succeeded in amplifying one copy of a circular DNA molecule more than 1012-fold to give microgram quantities of amplification product without using submicroliter reaction volumes. Furthermore, a ligation strategy was elaborated to circularize only the desired DNA sequence and eliminate undesired ligation-products. A combination of these methods was able to amplify and ligate a large construct without undesired DNA sequences and at microgram quantities within one day. Therefore, these methods have the possibility to improve DNA cloning techniques that have been restricted by the limitations of PCR methods or by the host cell.
Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers Hirokazu Takahashi 1, Kimiko Yamamoto 2, Toshio Ohtani 1, Shigeru Sugiyama 11, NanoBiotechnology Laboratory, Food Engineering Division, National Food Research Institute, National Agriculture and Food Research Organization, Ibaraki, Japan
2, Insect Genome Laboratory, National Institute of Agrobiological Sciences, Ibaraki, Japan
The predominant method for DNA cloning is by propagation in biological hosts, but this method has limitations because certain sequences are difficult to clone using any combination of available hosts or vectors. Recently, multiply-primed rolling circle amplification (MPRCA) has been applied to overcome the problems of the DNA cloning via host cells. However, when MPRCA is used to amplify from minute quantities of DNA template, the products are mostly by-product DNA molecules generated by false priming and primer dimer formation. This study demonstrates that MPRCA using random RNA primers—instead of DNA primers—blocked the synthesis of by-products and succeeded in amplifying one copy of a circular DNA molecule more than 1012-fold to give microgram quantities of amplification product without using submicroliter reaction volumes. Furthermore, a ligation strategy was elaborated to circularize only the desired DNA sequence and eliminate undesired ligation-products. A combination of these methods was able to amplify and ligate a large construct without undesired DNA sequences and at microgram quantities within one day. Therefore, these methods have the possibility to improve DNA cloning techniques that have been restricted by the limitations of PCR methods or by the host cell.
Saturday, July 04, 2009
Más Chistes
Un viejito va al médico a preguntarle si puede tener
hijos con su esposa de setenta. El médico le da un tarrito y le dice que le
traiga al día siguiente una muestra de semen.
Cuando vuelve, el viejito le dice:
- Doctor, no he podido traerle la muestra de semen.
- ¿Lo ha intentado?
- Sí, Doctor. Primero con la mano derecha, después con la izquierda.
Luego lo intentó mi esposa, primero con las dos manos y luego con los
dientes, pero no hubo forma...
¡No pudimos abrir el tarrito....!
MÁS DE VIEJITOS
El viejito se está bañando cuando de pronto siente
que su pene comienza a
endurecerse y llama a su viejita:
- ¡Josefina, Josefina, ven pronto!
La viejita va corriendo al baño y cuando ve este
espectáculo le dice al viejito:
¿Me desnudo, me desnudo? - pregunta con emoción la viejita.
- ¡Déjate de eso y ve rápido a buscar la cámara para tomar una foto!
EN MISA
El padre dice en la misa:
- Sí tienen fe se sanarán, pongan su mano sobre la parte afectada y el
milagro ocurrirá.
Una pareja de viejitos esta oyendo el sermón y el
viejito baja la mano con disimulo y la pone entre sus piernas. La viejita lo
ve y le dice:
- Viejo, el Cura dijo milagro, no resurrección.
¡CUÁNTAS CANAS!
Se desnudan el viejito y la viejita, y dice el
esposo todo sorprendido:
- ¡Ay, mi amor. Como tienes de canas allá abajo!
- No son canas, son telarañas, ¡viejo inútil!
EN TRATAMIENTO
Estaban en una reunión familiar y le dicen a los abuelos:
- Bueno, y ¿cómo va su vida sexual?
A lo cual la viejita contestó:
- Pues estamos en tratamiento.
- ¿Cómo así que en tratamiento?
- Pues sí: el trata y yo miento.
VISITA CONYUGAL
Llega una viejita a la cárcel el día de la visita conyugal y le dice al guardia
- Señor yo vengo a la visita conyugal.
- El guardia asombrado le pregunta: ¿Pero señora,
con quién?
- Con cualquiera, con cualquiera...
LA RANA Y EL VIEJO
Iba un viejito por el bosque cuando escuchó a sus
pies una débil voz.
Se agachó y descubrió que quien le hablaba era una
ranita:
- 'Soy una princesa hermosa, erótica y sensual,
diestra en todos los placeres de la carne y el amor.. La reina mala,
envidiosa de mis encantos, me convirtió en rana, pero si me das un beso,
volveré a ser quien era y te daré todos los goces y deleites que
mi voluptuoso temperamento y mi ardiente concupiscencia pueden producir'.
El viejito levanta la rana y se la echa en el bolsillo.
Asoma la cabeza la ranita y le pregunta muy desconcertada:
-¿Qué? ¿No me vas a besar?
- ¡No! -Respondió el viejecito
- A mi edad es más divertido tener una rana que habla, que una maniática sexual!!!!!!!!!!!!!!!!
hijos con su esposa de setenta. El médico le da un tarrito y le dice que le
traiga al día siguiente una muestra de semen.
Cuando vuelve, el viejito le dice:
- Doctor, no he podido traerle la muestra de semen.
- ¿Lo ha intentado?
- Sí, Doctor. Primero con la mano derecha, después con la izquierda.
Luego lo intentó mi esposa, primero con las dos manos y luego con los
dientes, pero no hubo forma...
¡No pudimos abrir el tarrito....!
MÁS DE VIEJITOS
El viejito se está bañando cuando de pronto siente
que su pene comienza a
endurecerse y llama a su viejita:
- ¡Josefina, Josefina, ven pronto!
La viejita va corriendo al baño y cuando ve este
espectáculo le dice al viejito:
¿Me desnudo, me desnudo? - pregunta con emoción la viejita.
- ¡Déjate de eso y ve rápido a buscar la cámara para tomar una foto!
EN MISA
El padre dice en la misa:
- Sí tienen fe se sanarán, pongan su mano sobre la parte afectada y el
milagro ocurrirá.
Una pareja de viejitos esta oyendo el sermón y el
viejito baja la mano con disimulo y la pone entre sus piernas. La viejita lo
ve y le dice:
- Viejo, el Cura dijo milagro, no resurrección.
¡CUÁNTAS CANAS!
Se desnudan el viejito y la viejita, y dice el
esposo todo sorprendido:
- ¡Ay, mi amor. Como tienes de canas allá abajo!
- No son canas, son telarañas, ¡viejo inútil!
EN TRATAMIENTO
Estaban en una reunión familiar y le dicen a los abuelos:
- Bueno, y ¿cómo va su vida sexual?
A lo cual la viejita contestó:
- Pues estamos en tratamiento.
- ¿Cómo así que en tratamiento?
- Pues sí: el trata y yo miento.
VISITA CONYUGAL
Llega una viejita a la cárcel el día de la visita conyugal y le dice al guardia
- Señor yo vengo a la visita conyugal.
- El guardia asombrado le pregunta: ¿Pero señora,
con quién?
- Con cualquiera, con cualquiera...
LA RANA Y EL VIEJO
Iba un viejito por el bosque cuando escuchó a sus
pies una débil voz.
Se agachó y descubrió que quien le hablaba era una
ranita:
- 'Soy una princesa hermosa, erótica y sensual,
diestra en todos los placeres de la carne y el amor.. La reina mala,
envidiosa de mis encantos, me convirtió en rana, pero si me das un beso,
volveré a ser quien era y te daré todos los goces y deleites que
mi voluptuoso temperamento y mi ardiente concupiscencia pueden producir'.
El viejito levanta la rana y se la echa en el bolsillo.
Asoma la cabeza la ranita y le pregunta muy desconcertada:
-¿Qué? ¿No me vas a besar?
- ¡No! -Respondió el viejecito
- A mi edad es más divertido tener una rana que habla, que una maniática sexual!!!!!!!!!!!!!!!!
Chistes
!! Como para quedarse mudo !!.
Después de 25 años de casados, un día miré a mi esposa y le dije:
Querida, ¿te das cuenta de que hace 25 años yo tenía un apartamento barato, un carro barato, dormía en un sofá-cama barato, veía televisión en un televisor de 10 pulgadas en blanco y negro, pero dormía todas las noches con una despampanante joven de 25 años? Y ahora tengo una hermosa casa, un carro último modelo, una cama grande, un televisor enorme de plasma, amen de otras delicadezas de vida.
Pero ahora..... duermo todas las noches con una mujer de CUARENTA Y TANTOS años con un poquito de sobrepeso.
! Me parece que no estás manejando bien tu parte de la situación, creo necesario, que pongas mas de tu parte !.
Mi esposa, a la que considero una persona muy razonable. Me dijo:
Mi amor, es procedente que salgas y busques una despampanante joven de 25 años, super delgada para que te acuestes con ella y seas mas feliz, que yo por mi parte solo me aseguraré de que... vuelvas a vivir en un apartamento barato, tengas un carro barato y duermas en un sofá-cama barato... ¡ Todos seremos felices !
MORALEJA:
LAS MUJERES SON Y SERÁN SIEMPRE MAS INTELIGENTES QUE BONITAS.
LOS HOMBRES SON Y SERÁN SIEMPRE MAS CALIENTES Y PENDEJOS QUE INTELIGENTES.
!!!!!!!!!!
Después de 25 años de casados, un día miré a mi esposa y le dije:
Querida, ¿te das cuenta de que hace 25 años yo tenía un apartamento barato, un carro barato, dormía en un sofá-cama barato, veía televisión en un televisor de 10 pulgadas en blanco y negro, pero dormía todas las noches con una despampanante joven de 25 años? Y ahora tengo una hermosa casa, un carro último modelo, una cama grande, un televisor enorme de plasma, amen de otras delicadezas de vida.
Pero ahora..... duermo todas las noches con una mujer de CUARENTA Y TANTOS años con un poquito de sobrepeso.
! Me parece que no estás manejando bien tu parte de la situación, creo necesario, que pongas mas de tu parte !.
Mi esposa, a la que considero una persona muy razonable. Me dijo:
Mi amor, es procedente que salgas y busques una despampanante joven de 25 años, super delgada para que te acuestes con ella y seas mas feliz, que yo por mi parte solo me aseguraré de que... vuelvas a vivir en un apartamento barato, tengas un carro barato y duermas en un sofá-cama barato... ¡ Todos seremos felices !
MORALEJA:
LAS MUJERES SON Y SERÁN SIEMPRE MAS INTELIGENTES QUE BONITAS.
LOS HOMBRES SON Y SERÁN SIEMPRE MAS CALIENTES Y PENDEJOS QUE INTELIGENTES.
!!!!!!!!!!
Thursday, May 14, 2009
अर्तिक्लेस फॉर मुटेशन
http://nar.oxfordjournals.org/cgi/content/abstract/29/14/e71
Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection
Piotr Kozlowski and Wlodzimierz J. Krzyzosiak*
Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
SSCP and heteroduplex analysis (HA) continue to be the most popular methods of mutation detection due to their simplicity, high sensitivity and low cost. The advantages of these methods are most clearly visible when large genes, such as BRCA1 and BRCA2, are scanned for scattered unknown mutations and/or when a large number of DNA samples is screened for specific mutations. Here we describe a novel combined SSCP/duplex analysis adapted to the modern capillary electrophoresis (CE) system, which takes advantage of multicolor labeling of DNA fragments and laser-induced fluorescence detection. In developing this method, we first established the optimum conditions for homoduplex and heteroduplex analysis by CE. These were determined based on comprehensive analysis of representative Tamra-500 markers and BRCA1 fragments at different concentrations of sieving polymer and temperatures in the presence or absence of glycerol. The intrinsic features of DNA duplex structures are discussed in detail to explain differences in the migration rates between various types of duplexes. When combined SSCP/duplex analysis was carried out in single conditions, those found to be optimal for analysis of duplexes, all 31 BRCA1 and BRCA2 mutations, polymorphisms and variants tested were detected. It is worth noting that the panel of analyzed sequence variants was enriched in base substitutions, which are usually more difficult to detect. The sensitivity of mutation detection in the SSCP portion alone was 90%, and that in the duplex portion was 81% in the single conditions of electrophoresis. As is also shown here, the proposed combined SSCP/duplex analysis by CE has the potential of being applied to the analysis of pooled genomic DNA samples, and to multiplex analysis of amplicons from different gene fragments. These modifications may further reduce the costs of analysis, making the method attractive for large scale application in SNP scanning and screening.
* To whom correspondence should be addressed. Tel: +48 61 852 8503; Fax: +48 61 852 0532; Email:
http://www.clinchem.org/cgi/content/abstract/47/2/164
Enabling Large-Scale Pharmacogenetic Studies by High-Throughput Mutation Detection and Genotyping Technologies
Michael M. Shi1
1 Department of Applied Genomics, Genometrix Inc., The Woodlands, TX 77381, and Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109.
Abstract
Background: Pharmacogenetics is a scientific discipline that examines the genetic basis for individual variations in response to therapeutics. Pharmacogenetics promises to develop individualized medicines tailored to patients’ genotypes. However, identifying and genotyping a vast number of genetic polymorphisms in large populations also pose a great challenge.
Approach: This article reviews the recent technology development in mutation detection and genotyping with a focus on genotyping of single nucleotide polymorphisms (SNPs).
Content: Novel mutations/polymorphisms are commonly identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. With a large amount of public sequence information available, in silico SNP mapping has also emerged as a cost-efficient way for new polymorphism identification. Gel electrophoresis-based genotyping methods for known polymorphisms include PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing. Fluorescent dye-based genotyping technologies are emerging as high-throughput genotyping platforms, including oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan®, and molecular beacon genotyping. Rolling circle amplification and InvaderTM assays are able to genotype directly from genomic DNA without PCR amplification. DNA chip-based microarray and mass spectrometry genotyping technologies are the latest development in the genotyping arena.
Summary: Large-scale genotyping is crucial to the identification of the genetic make-ups that underlie the onset of diseases and individual variations in drug responses. Enabling technologies to identify genetic polymorphisms rapidly, accurately, and cost effectively will dramatically impact future drug and development processes.
http://www.nature.com/ng/journal/v18/n2/abs/ng0298-192.html
Mutation detection and typing of polymorphic loci through double-strand conformation analysis
J. Rafael Argüello1, Ann-Margaret Little1, 2, Andrea L. Pay1, David Gallardo1, Isabel Rojas1, Steven G.E. Marsh1, John M. Goldman1, 3 & J. Alejandro Madrigal1, 2, 3, 4
1Anthony Nolan Research Institute, The Royal Free Hospital, Pond Street, London, NW3 2QG, UK.
2Department of Haematology, The Royal Free Hospital, Pond Street, London, NW3 2QG, UK.
3Department of Haematology, The Imperial College School of Medicine, Du Cane Road, London, W12 ONN, UK.
4e-mail: Madrigal@RFHSM. ac.uk
Variations, such as nucleotide substitutions, deletions and insertions, within genes can affect the function of the gene product and in some cases be deleterious. Screening for known allelic variation is important for determining disease and gene associations1. Techniques which target specific mutations such as restriction enzyme polymorphism and oligonucleotide probe or PCR primer reactivity are useful for the detection of specific mutations, but these techniques are not generally effective for the identification of new mutations. Approaches for measuring changes in DNA conformation have been developed, based on the principle that DNA fragments which differ in nucleotide composition exhibit different mobilities after separation by polyacrylamide gel electrophoresis (PAGE; refs 2,3). Here we describe a conformation-based mutation detection system, double-strand conformation analysis (DSCA), which provides a simple means to detect genetic variants and to type complex polymorphic loci. We demonstrate the application of DSCA to detect genetic polymorphisms such as a single-nucleotide difference within DNA fragments of up to 979 base pairs in length. We present the application of DSCA in detecting four different mutations in the cystic fibrosis gene (CFTR) and 131 different alleles encoded by HLA class I genes.
http://genome.cshlp.org/content/10/9/1403.full
Rapid Detection of Deletion, Insertion, and Substitution Mutations via Heteroduplex Analysis Using Capillary- and Microchip-Based Electrophoresis
Huijun Tian1, Lawrence C. Brody2, and James P. Landers3,4
+Author Affiliations
1Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA; 2Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; 3Department of Chemistry and Department of Pathology, University of Virginia, Charlottesville, Virginia 22901, USA
Next SectionAbstract
In this report, we explore the potential of capillary and microchip electrophoresis for heteroduplex analysis– (HDA) based mutation detection. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify the DNA fragments ranging from 130 to 400 bp. The effects of DNA fragment length, matrix additives, pH, and salt were evaluated for capillary electrophoresis– (CE) and/or microchip electrophoresis–based HDA, using six heterozygous mutations,185delAG, E1250X (3867GT), R1443G(4446CG), 5382insC, 5677insA inBRCA1, and 6174delT in BRCA2. For this system, the effective fragment size for CE-based HDA was found in the range of 200–300 bp, however, the effective range was 150–260 bp for microchip-based HDA. Sensitivity studies show CE-based HDA could detect a mutated DNA present at only 1%–10% of the total DNA. Discrimination between wild-type and deletion or insertion mutations in BRCA1 and BRCA2 with CE-based HDA could be achieved in <8 min, while the substitution mutations required 14 min of analysis time. For each mutation region, 15 samples were run to confirm the accuracy and reproducibility of the method. Using the method described, two previously reported mutations, E1038G (3232AG, missense) and 4427 C/T (4427CT, polymorphism), were detected in the tested samples and confirmed by DNA sequencing. Translation of the CE-based methodology to the microchip format allowed the analysis time for each mutation to be decreased to 130 sec. Based on the results obtained with this model system, it is possible that CE-based HDA methodologies can be developed and used effectively in genetic testing. The fast separation time and automated operation afforded with CE instrumentation provide a powerful system for screening mutations that include small deletions, insertions, and point mutations. Translation to the microchip platform, especially to a multichannel microchip system, would allow for screening mutations with high throughput.
Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection
Piotr Kozlowski and Wlodzimierz J. Krzyzosiak*
Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland
SSCP and heteroduplex analysis (HA) continue to be the most popular methods of mutation detection due to their simplicity, high sensitivity and low cost. The advantages of these methods are most clearly visible when large genes, such as BRCA1 and BRCA2, are scanned for scattered unknown mutations and/or when a large number of DNA samples is screened for specific mutations. Here we describe a novel combined SSCP/duplex analysis adapted to the modern capillary electrophoresis (CE) system, which takes advantage of multicolor labeling of DNA fragments and laser-induced fluorescence detection. In developing this method, we first established the optimum conditions for homoduplex and heteroduplex analysis by CE. These were determined based on comprehensive analysis of representative Tamra-500 markers and BRCA1 fragments at different concentrations of sieving polymer and temperatures in the presence or absence of glycerol. The intrinsic features of DNA duplex structures are discussed in detail to explain differences in the migration rates between various types of duplexes. When combined SSCP/duplex analysis was carried out in single conditions, those found to be optimal for analysis of duplexes, all 31 BRCA1 and BRCA2 mutations, polymorphisms and variants tested were detected. It is worth noting that the panel of analyzed sequence variants was enriched in base substitutions, which are usually more difficult to detect. The sensitivity of mutation detection in the SSCP portion alone was 90%, and that in the duplex portion was 81% in the single conditions of electrophoresis. As is also shown here, the proposed combined SSCP/duplex analysis by CE has the potential of being applied to the analysis of pooled genomic DNA samples, and to multiplex analysis of amplicons from different gene fragments. These modifications may further reduce the costs of analysis, making the method attractive for large scale application in SNP scanning and screening.
* To whom correspondence should be addressed. Tel: +48 61 852 8503; Fax: +48 61 852 0532; Email:
http://www.clinchem.org/cgi/content/abstract/47/2/164
Enabling Large-Scale Pharmacogenetic Studies by High-Throughput Mutation Detection and Genotyping Technologies
Michael M. Shi1
1 Department of Applied Genomics, Genometrix Inc., The Woodlands, TX 77381, and Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109.
Abstract
Background: Pharmacogenetics is a scientific discipline that examines the genetic basis for individual variations in response to therapeutics. Pharmacogenetics promises to develop individualized medicines tailored to patients’ genotypes. However, identifying and genotyping a vast number of genetic polymorphisms in large populations also pose a great challenge.
Approach: This article reviews the recent technology development in mutation detection and genotyping with a focus on genotyping of single nucleotide polymorphisms (SNPs).
Content: Novel mutations/polymorphisms are commonly identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. With a large amount of public sequence information available, in silico SNP mapping has also emerged as a cost-efficient way for new polymorphism identification. Gel electrophoresis-based genotyping methods for known polymorphisms include PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing. Fluorescent dye-based genotyping technologies are emerging as high-throughput genotyping platforms, including oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan®, and molecular beacon genotyping. Rolling circle amplification and InvaderTM assays are able to genotype directly from genomic DNA without PCR amplification. DNA chip-based microarray and mass spectrometry genotyping technologies are the latest development in the genotyping arena.
Summary: Large-scale genotyping is crucial to the identification of the genetic make-ups that underlie the onset of diseases and individual variations in drug responses. Enabling technologies to identify genetic polymorphisms rapidly, accurately, and cost effectively will dramatically impact future drug and development processes.
http://www.nature.com/ng/journal/v18/n2/abs/ng0298-192.html
Mutation detection and typing of polymorphic loci through double-strand conformation analysis
J. Rafael Argüello1, Ann-Margaret Little1, 2, Andrea L. Pay1, David Gallardo1, Isabel Rojas1, Steven G.E. Marsh1, John M. Goldman1, 3 & J. Alejandro Madrigal1, 2, 3, 4
1Anthony Nolan Research Institute, The Royal Free Hospital, Pond Street, London, NW3 2QG, UK.
2Department of Haematology, The Royal Free Hospital, Pond Street, London, NW3 2QG, UK.
3Department of Haematology, The Imperial College School of Medicine, Du Cane Road, London, W12 ONN, UK.
4e-mail: Madrigal@RFHSM. ac.uk
Variations, such as nucleotide substitutions, deletions and insertions, within genes can affect the function of the gene product and in some cases be deleterious. Screening for known allelic variation is important for determining disease and gene associations1. Techniques which target specific mutations such as restriction enzyme polymorphism and oligonucleotide probe or PCR primer reactivity are useful for the detection of specific mutations, but these techniques are not generally effective for the identification of new mutations. Approaches for measuring changes in DNA conformation have been developed, based on the principle that DNA fragments which differ in nucleotide composition exhibit different mobilities after separation by polyacrylamide gel electrophoresis (PAGE; refs 2,3). Here we describe a conformation-based mutation detection system, double-strand conformation analysis (DSCA), which provides a simple means to detect genetic variants and to type complex polymorphic loci. We demonstrate the application of DSCA to detect genetic polymorphisms such as a single-nucleotide difference within DNA fragments of up to 979 base pairs in length. We present the application of DSCA in detecting four different mutations in the cystic fibrosis gene (CFTR) and 131 different alleles encoded by HLA class I genes.
http://genome.cshlp.org/content/10/9/1403.full
Rapid Detection of Deletion, Insertion, and Substitution Mutations via Heteroduplex Analysis Using Capillary- and Microchip-Based Electrophoresis
Huijun Tian1, Lawrence C. Brody2, and James P. Landers3,4
+Author Affiliations
1Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA; 2Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; 3Department of Chemistry and Department of Pathology, University of Virginia, Charlottesville, Virginia 22901, USA
Next SectionAbstract
In this report, we explore the potential of capillary and microchip electrophoresis for heteroduplex analysis– (HDA) based mutation detection. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify the DNA fragments ranging from 130 to 400 bp. The effects of DNA fragment length, matrix additives, pH, and salt were evaluated for capillary electrophoresis– (CE) and/or microchip electrophoresis–based HDA, using six heterozygous mutations,185delAG, E1250X (3867GT), R1443G(4446CG), 5382insC, 5677insA inBRCA1, and 6174delT in BRCA2. For this system, the effective fragment size for CE-based HDA was found in the range of 200–300 bp, however, the effective range was 150–260 bp for microchip-based HDA. Sensitivity studies show CE-based HDA could detect a mutated DNA present at only 1%–10% of the total DNA. Discrimination between wild-type and deletion or insertion mutations in BRCA1 and BRCA2 with CE-based HDA could be achieved in <8 min, while the substitution mutations required 14 min of analysis time. For each mutation region, 15 samples were run to confirm the accuracy and reproducibility of the method. Using the method described, two previously reported mutations, E1038G (3232AG, missense) and 4427 C/T (4427CT, polymorphism), were detected in the tested samples and confirmed by DNA sequencing. Translation of the CE-based methodology to the microchip format allowed the analysis time for each mutation to be decreased to 130 sec. Based on the results obtained with this model system, it is possible that CE-based HDA methodologies can be developed and used effectively in genetic testing. The fast separation time and automated operation afforded with CE instrumentation provide a powerful system for screening mutations that include small deletions, insertions, and point mutations. Translation to the microchip platform, especially to a multichannel microchip system, would allow for screening mutations with high throughput.
http://jmd.amjpathol.org/cgi/content/abstract/4/2/103
Competitive Amplification and Unspecific Amplification in Polymerase Chain Reaction with Confronting Two-Pair Primers
Nobuyuki Hamajima*, Toshiko Saito*, Keitaro Matsuo* and Kazuo Tajima*
From the Division of Epidemiology and Prevention, * Aichi Cancer Center Research Institute, Nagoya; and Nagoya University Graduate School of Medicine, Nagoya, Japan
Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is an inexpensive, time-saving genotyping method that is applicable for most single nucleotide polymorphisms. To date, we have applied PCR-CTPP successfully for the genotyping of more than 30 polymorphisms. This paper demonstrates the differences in DNA amplification among different annealing temperatures of PCR-CTPP with given melting temperatures for four primers. The NQO1 C609T (Pro187Ser) polymorphism was used as an example. Two sets of four primers were applied for PCR-CTPP; the first set with different melting temperatures (Tms), and the second with similar Tms. The comparisons with one-pair primer PCR (allele-specific PCR) revealed that PCR-CTPP amplified DNA more specifically than allele-specific PCR. The primers with different Tms caused competitive DNA amplification for heterozygous genotype. Four primers with similar Tms amplified both alleles unspecifically at a lower annealing temperature, while the same DNA samples were correctly genotyped under an optimal annealing temperature. These findings are unique for PCR-CTPP, and important characteristics when the primers and annealing temperatures in PCR-CTPP are designed. The knowledge of these characteristics will extend the applicability of PCR-CTPP for polymorphism genotyping.
Competitive Amplification and Unspecific Amplification in Polymerase Chain Reaction with Confronting Two-Pair Primers
Nobuyuki Hamajima*, Toshiko Saito*, Keitaro Matsuo* and Kazuo Tajima*
From the Division of Epidemiology and Prevention, * Aichi Cancer Center Research Institute, Nagoya; and Nagoya University Graduate School of Medicine, Nagoya, Japan
Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is an inexpensive, time-saving genotyping method that is applicable for most single nucleotide polymorphisms. To date, we have applied PCR-CTPP successfully for the genotyping of more than 30 polymorphisms. This paper demonstrates the differences in DNA amplification among different annealing temperatures of PCR-CTPP with given melting temperatures for four primers. The NQO1 C609T (Pro187Ser) polymorphism was used as an example. Two sets of four primers were applied for PCR-CTPP; the first set with different melting temperatures (Tms), and the second with similar Tms. The comparisons with one-pair primer PCR (allele-specific PCR) revealed that PCR-CTPP amplified DNA more specifically than allele-specific PCR. The primers with different Tms caused competitive DNA amplification for heterozygous genotype. Four primers with similar Tms amplified both alleles unspecifically at a lower annealing temperature, while the same DNA samples were correctly genotyped under an optimal annealing temperature. These findings are unique for PCR-CTPP, and important characteristics when the primers and annealing temperatures in PCR-CTPP are designed. The knowledge of these characteristics will extend the applicability of PCR-CTPP for polymorphism genotyping.
http://nar.oxfordjournals.org/cgi/content/abstract/29/17/e88
An efficient procedure for genotyping single nucleotide polymorphisms
Shu Ye*, Sahar Dhillon, Xiayi Ke, Andrew R. Collins and Ian N. M. Day
Human Genetics Research Division, University of Southampton, Duthie Building (MP808), Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK
Analysis of single nucleotide polymorphisms (SNPs) has been and will be increasingly utilized in various genetic disciplines, particularly in studying genetic determinants of complex diseases. Such studies will be facilitated by rapid, simple, low cost and high throughput methodologies for SNP genotyping. One such method is reported here, named tetra-primer ARMS-PCR, which employs two primer pairs to amplify, respectively, the two different alleles of a SNP in a single PCR reaction. A computer program for designing primers was developed. Tetra-primer ARMS-PCR was combined with microplate array diagonal gel electrophoresis, gaining the advantage of high throughput for gel-based resolution of tetra-primer ARMS-PCR products. The technique was applied to analyse a number of SNPs and the results were completely consistent with those from an independent method, restriction fragment length polymorphism analysis.
* To whom correspondence should be addressed. Tel: +44 23 8079 4929
--------------------------------------------------------------------------------
C. A. Milbury, J. Li, and G. M. Makrigiorgos
PCR-Based Methods for the Enrichment of Minority Alleles and Mutations
Clin. Chem., April 1, 2009; 55(4): 632 - 640.
[Abstract] [Full Text] [PDF]
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J. Li, L. Wang, P. A. Janne, and G. M. Makrigiorgos
Coamplification at Lower Denaturation Temperature-PCR Increases Mutation-Detection Selectivity of TaqMan-Based Real-Time PCR
Clin. Chem., April 1, 2009; 55(4): 748 - 756.
[Abstract] [Full Text] [PDF]
An efficient procedure for genotyping single nucleotide polymorphisms
Shu Ye*, Sahar Dhillon, Xiayi Ke, Andrew R. Collins and Ian N. M. Day
Human Genetics Research Division, University of Southampton, Duthie Building (MP808), Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK
Analysis of single nucleotide polymorphisms (SNPs) has been and will be increasingly utilized in various genetic disciplines, particularly in studying genetic determinants of complex diseases. Such studies will be facilitated by rapid, simple, low cost and high throughput methodologies for SNP genotyping. One such method is reported here, named tetra-primer ARMS-PCR, which employs two primer pairs to amplify, respectively, the two different alleles of a SNP in a single PCR reaction. A computer program for designing primers was developed. Tetra-primer ARMS-PCR was combined with microplate array diagonal gel electrophoresis, gaining the advantage of high throughput for gel-based resolution of tetra-primer ARMS-PCR products. The technique was applied to analyse a number of SNPs and the results were completely consistent with those from an independent method, restriction fragment length polymorphism analysis.
* To whom correspondence should be addressed. Tel: +44 23 8079 4929
--------------------------------------------------------------------------------
C. A. Milbury, J. Li, and G. M. Makrigiorgos
PCR-Based Methods for the Enrichment of Minority Alleles and Mutations
Clin. Chem., April 1, 2009; 55(4): 632 - 640.
[Abstract] [Full Text] [PDF]
--------------------------------------------------------------------------------
J. Li, L. Wang, P. A. Janne, and G. M. Makrigiorgos
Coamplification at Lower Denaturation Temperature-PCR Increases Mutation-Detection Selectivity of TaqMan-Based Real-Time PCR
Clin. Chem., April 1, 2009; 55(4): 748 - 756.
[Abstract] [Full Text] [PDF]
http://nar.oxfordjournals.org/cgi/content/abstract/35/12/e84
s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis
Jin Li1, Ross Berbeco2, Robert J. Distel3, Pasi A. Jänne4, Lilin Wang1 and G. Mike Makrigiorgos1,2,*
1Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA, 2Physics and Department of Medical Oncology, Department of Radiation Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA, 3Translational Research Laboratory: Center for Clinical and Translational Research, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA and 4Lowe Center for Thoracic Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA
*To whom correspondence should be addressed. Tel: +1-617-525-7122; Fax: +1-617-587-6037; Email: mmakrigiorgos@partners.org
Received March 1, 2007. Revised May 2, 2007. Accepted May 2, 2007.
The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (SurveyorTM) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine.
s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis
Jin Li1, Ross Berbeco2, Robert J. Distel3, Pasi A. Jänne4, Lilin Wang1 and G. Mike Makrigiorgos1,2,*
1Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA, 2Physics and Department of Medical Oncology, Department of Radiation Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA, 3Translational Research Laboratory: Center for Clinical and Translational Research, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA and 4Lowe Center for Thoracic Oncology, Dana Farber-Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA, USA
*To whom correspondence should be addressed. Tel: +1-617-525-7122; Fax: +1-617-587-6037; Email: mmakrigiorgos@partners.org
Received March 1, 2007. Revised May 2, 2007. Accepted May 2, 2007.
The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (SurveyorTM) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine.
http://nar.oxfordjournals.org/cgi/content/abstract/34/13/e99
High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling
Bradley J. Till1,*, Troy Zerr1, Elisabeth Bowers1, Elizabeth A. Greene1, Luca Comai2 and Steven Henikoff1
1 Basic Sciences Division, Fred Hutchinson Cancer Research Center Seattle, WA 98109, USA 2 Department of Biology, University of Washington Seattle, WA 98195, USA
*To whom correspondence should be addressed. Tel: +1 206 685 1949; Fax: +1 206 616 2011; Email: btill@fhcrc.org
Received May 26, 2006. Revised June 21, 2006. Accepted June 21, 2006.
Human individuals differ from one another at only 0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease.
High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling
Bradley J. Till1,*, Troy Zerr1, Elisabeth Bowers1, Elizabeth A. Greene1, Luca Comai2 and Steven Henikoff1
1 Basic Sciences Division, Fred Hutchinson Cancer Research Center Seattle, WA 98109, USA 2 Department of Biology, University of Washington Seattle, WA 98195, USA
*To whom correspondence should be addressed. Tel: +1 206 685 1949; Fax: +1 206 616 2011; Email: btill@fhcrc.org
Received May 26, 2006. Revised June 21, 2006. Accepted June 21, 2006.
Human individuals differ from one another at only 0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease.
http://crop.scijournals.org/cgi/content/abstract/49/2/381
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
CEL I Nuclease Digestion for SNP Discovery and Marker Development in Common Bean (Phaseolus vulgaris L.)
Carlos H. Galeano, Marcela Gomez, Lina M. Rodriguez and Matthew W. Blair*
CIAT- International Center for Tropical Agriculture, A.A. 6713, Cali, Colombia, South America
* Corresponding author (m.blair@cgiar.org).
Single nucleotide polymorphisms (SNPs) are the most common sequence difference found in plant genomes, yet they have not been widely exploited for producing molecular markers in common bean (Phaseolus vulgaris L.). The objective of this study was to develop a SNP assay based on a type of heteroduplex mismatch cleavage called EcoTILLING for molecular marker development in this important legume, and apply the assay (i) to the conversion of a sequence-characterized amplified region (SCAR) marker useful for selecting virus resistance (SR2) and (ii) to the screening of SNP polymorphisms in newly developed expressed sequence tag (EST)–based markers. The SNP assay involved heteroduplex mismatch cleavage by a single-strand specific nuclease ‘CEL I’ which was used to uncover two SNPs in the SR2 fragment and 22 SNPs in 37 candidate ESTs, some of which were used in segregation analysis. While developing the SNP techniques we tested several platforms, including LI-COR, nondenaturing polyacrylamide, and agarose gel detection. The agarose gel system was used for SNP genetic mapping in two common bean mapping populations, showing that heteroduplex cleavage is a useful technique for increasing molecular marker number for the crop. Examples are given of mapped SNP markers for the phytic acid pathway gene for myo-inositol-1-phosphate synthase and a drought tolerance–related gene, S-adenosylmethionine decarboxylase.
Abbreviations: ARMS, amplification refractory mutation system • BGYMV, bean golden yellow mosaic virus • CAPS, cleaved amplified polymorphic sequence • COS, conserved ortholog set • dHPLC, denaturing high performance liquid chromatography • EST, expressed sequence tag • SCAR • sequence-characterized amplified region • SNP, single nucleotide polymorphism • SSCP, single strand conformational polymorphism • RIL, recombinant inbred line • TILLING, target induced local lesion in genomes
J. Li, R. Berbeco, R. J. Distel, P. A. Janne, L. Wang, and G. M. Makrigiorgos
s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis
Nucleic Acids Res., June 9, 2007; 35(12): e84 - e84.
http://bfgp.oxfordjournals.org/cgi/content/abstract/eln046v1
http://bfgp.oxfordjournals.org/cgi/content/abstract/eln046v1
Reverse genetics in zebrafish by TILLING
Cecilia B. Moens, Thomas M. Donn, Emma R. Wolf-Saxon and Taylur P. Ma
Corresponding author. Cecilia B. Moens, HHMI and Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Ave. N., Seattle, WA 98109, USA. Tel: +1 206 667 5627; Fax: +1 206 667 5939; E-mail: cmoens@fhcrc.org
TILLING, for Targeting Induced Local Lesions in Genomes, is a reverse genetics strategy that identifies mutations in specific genes of interest in chemically mutagenized populations. First described in 2000 for mutation detection in Arabidopsis, TILLING is now used in a wide range of plants including soybean, rice, barley and maize as well as for animal model systems, including Arabidopsis, Drosophila, Caenorhabditis elegans, rat, medaka and zebrafish and for the discovery of naturally occurring polymorphisms in humans. This review summarizes current TILLING methodologies as they have been applied to the zebrafish, ongoing TILLING projects and resources in the zebrafish community, and the future of zebrafish TILLING.
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
CEL I Nuclease Digestion for SNP Discovery and Marker Development in Common Bean (Phaseolus vulgaris L.)
Carlos H. Galeano, Marcela Gomez, Lina M. Rodriguez and Matthew W. Blair*
CIAT- International Center for Tropical Agriculture, A.A. 6713, Cali, Colombia, South America
* Corresponding author (m.blair@cgiar.org).
Single nucleotide polymorphisms (SNPs) are the most common sequence difference found in plant genomes, yet they have not been widely exploited for producing molecular markers in common bean (Phaseolus vulgaris L.). The objective of this study was to develop a SNP assay based on a type of heteroduplex mismatch cleavage called EcoTILLING for molecular marker development in this important legume, and apply the assay (i) to the conversion of a sequence-characterized amplified region (SCAR) marker useful for selecting virus resistance (SR2) and (ii) to the screening of SNP polymorphisms in newly developed expressed sequence tag (EST)–based markers. The SNP assay involved heteroduplex mismatch cleavage by a single-strand specific nuclease ‘CEL I’ which was used to uncover two SNPs in the SR2 fragment and 22 SNPs in 37 candidate ESTs, some of which were used in segregation analysis. While developing the SNP techniques we tested several platforms, including LI-COR, nondenaturing polyacrylamide, and agarose gel detection. The agarose gel system was used for SNP genetic mapping in two common bean mapping populations, showing that heteroduplex cleavage is a useful technique for increasing molecular marker number for the crop. Examples are given of mapped SNP markers for the phytic acid pathway gene for myo-inositol-1-phosphate synthase and a drought tolerance–related gene, S-adenosylmethionine decarboxylase.
Abbreviations: ARMS, amplification refractory mutation system • BGYMV, bean golden yellow mosaic virus • CAPS, cleaved amplified polymorphic sequence • COS, conserved ortholog set • dHPLC, denaturing high performance liquid chromatography • EST, expressed sequence tag • SCAR • sequence-characterized amplified region • SNP, single nucleotide polymorphism • SSCP, single strand conformational polymorphism • RIL, recombinant inbred line • TILLING, target induced local lesion in genomes
J. Li, R. Berbeco, R. J. Distel, P. A. Janne, L. Wang, and G. M. Makrigiorgos
s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis
Nucleic Acids Res., June 9, 2007; 35(12): e84 - e84.
http://bfgp.oxfordjournals.org/cgi/content/abstract/eln046v1
http://bfgp.oxfordjournals.org/cgi/content/abstract/eln046v1
Reverse genetics in zebrafish by TILLING
Cecilia B. Moens, Thomas M. Donn, Emma R. Wolf-Saxon and Taylur P. Ma
Corresponding author. Cecilia B. Moens, HHMI and Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Ave. N., Seattle, WA 98109, USA. Tel: +1 206 667 5627; Fax: +1 206 667 5939; E-mail: cmoens@fhcrc.org
TILLING, for Targeting Induced Local Lesions in Genomes, is a reverse genetics strategy that identifies mutations in specific genes of interest in chemically mutagenized populations. First described in 2000 for mutation detection in Arabidopsis, TILLING is now used in a wide range of plants including soybean, rice, barley and maize as well as for animal model systems, including Arabidopsis, Drosophila, Caenorhabditis elegans, rat, medaka and zebrafish and for the discovery of naturally occurring polymorphisms in humans. This review summarizes current TILLING methodologies as they have been applied to the zebrafish, ongoing TILLING projects and resources in the zebrafish community, and the future of zebrafish TILLING.
http://www.clinchem.org/cgi/content/full/50/4/773
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Clinical Chemistry 50: 773-775, 2004; 10.1373/clinchem.2003.030445
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Technical Briefs
Detection of Five Common CFTR Mutations by Rapid-Cycle Real-Time Amplification Refractory Mutation System PCR
Eugene Dempsey1, David E. Barton2 and Fergus Ryan1,a
1 Department of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland;2 National Centre for Medical Genetics and Department of Paediatrics, University College Dublin, Our Lady’s Hospital for Sick Children, Crumlin, Dublin, Ireland
aaddress correspondence to this author at: Department of Biological Sciences, Dublin Institute of Technology, Kevin St., Dublin 8, Ireland; e-mail fergus.x.ryan@dit.ie
Cystic fibrosis is the most common autosomal recessive disease in Caucasian populations and has a carrier frequency of 1 in 25 (1). The gene involved codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane-associated protein involved in ion transport across the plasma membrane of epithelial cells. To date more than 1000 mutations have been described in this gene, and most are rare (2). By focusing on five common mutations it is possible to detect the disease-causing mutation in 90% of Irish patients (3). The five mutations [and the percentages of Irish (3) and worldwide (2) cases] are F508del (77.4%, 66.0%), G551D (7.1%, 1.6%), R117H (2.7%, 0.3%), 621+1 G>T (1.4%, 0.7%), and G542X (0.5%, 2.4%). Four of these fall into the severe class of mutations in which the mRNA is incorrectly spliced (621+1 G>T), or in which the protein is not synthesized (G542X) or is blocked during processing (F508del), or its regulation is blocked (G551D).
Mutations in the CFTR gene can be detected by many mutation-detection systems, including single-strand conformation polymorphism analysis (4), restriction fragment length polymorphism analysis, Amplification Refractory Mutation System (ARMS) PCR (5), and more recently, real-time PCR systems using Sybr Green 1 (6) or hybridization probes (7). Currently, for mutation detection using hybridization probes on the LightCycler system, the detector probe is designed to overlie the possible site of the mutation. This can have drawbacks when multiplexing in the same detection channel because peaks can become merged, making genotyping of a sample difficult.
This study aimed to improve the multiplexing capabilities of real-time PCR in its use for mutation detection. To this end we used ARMS PCR primers to selectively amplify the wild-type or mutant alleles in separate reactions. Subsequent detection of PCR products was carried out with a common set of hybridization probes. The advantage of this system is that it increases flexibility in design of the hybridization probe, allowing probes with precise melting temperatures (Tms) to be generated. In this way melting curve peaks can be at a predetermined Tm for a particular PCR product, which allows for easier multiplexing of real-time PCR. Similar ARMS-based techniques have been developed for the ABI Prism 7700 sequence detector from PE Applied Biosystems using TaqMan® probes (8). These protocols depend on the detection of an amplification curve and are limited to the detection of one mutation per color channel. Our protocol detects melting curve peaks post amplification and allows multiple mutations to be detected per color channel.
Both control samples for protocol optimization and test samples for the blinded study were obtained from the National Centre for Medical Genetics, Our Lady’s Hospital for Sick Children (Crumlin, Dublin, Ireland). This study has received ethical approval from the DIT ethical review board (Project Ref. SF01/02).
Two multiplex PCR reactions were optimized by use of previously published ARMS primers (5). The first reaction detects the G551D, R117H, and F508del mutations (87.2% frequency in Ireland, 67.9% worldwide). The F508del ARMS primers (CF-DFjN and CF-DFwM) are specific for this mutation and are not influenced by the benign I506V and F508C mutations. Because the CF-DFwM primer is complementary to the sequence distal to F508del, the presence of the I507del mutation is not recognized (5).
The second reaction detects the 621+1 G>T and G542X mutations (1.9% frequency in Ireland, 3.1% worldwide). A PCR product should be formed in both the A and B ARMS reactions regardless of the sample genotype.
Three sets of fluorescence resonance energy transfer hybridization probes were designed to detect the 10 different ARMS PCR products. R117H and 621+1 G>T ARMS products are detectable by the CF4ARMS-A (5'-CCTTTTGTAGGAAGTCACCAAAGCAGTAC-F-3') and CF4ARMS-P (5'-LCRed640-GCCTCTCTTACTGGGAAGAATCA-P-3') hybridization probes (F indicates a fluorescein, and P indicates phosphate). The F508del ARMS products are detected by the CF10ARMS-A (5'-GCACAGTGGAAGAATTTCATTCTGTTCTCAG-F-3') and CF10ARMS-P (5'-LCRed640-TTCCTTGGATTATGCCT-P-3') hybridization probes. The CF11ARMS-A (5'-TATGATTACATTAGAAGGAAGATGTGCCTTT-F-3') and CF11ARMS-P (5'-LCRed705-AATTCAGATTGAGCATACT-P-3') hybridization probes detect both the G551D and G542X ARMS products. The predicted Tms of the detector probes were calculated by use of MeltCalc software (www.meltcalc.com) (9). All probes and primers were synthesized by Proligo, France.
ARMS PCR was carried out on a LightCycler (Roche Diagnostics). The total reaction volume was 10 µL, containing 1 µL of 10x LightCycler DNA Master Hybridization enzyme mixture (Roche Diagnostics), 3 mM MgCl2, 0.1 µM hybridization probes, and ARMS primers concentrations as outlined in Table 1 . ARMS PCR and subsequent melting curve analysis were carried out as follows:
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Table 1. Concentrations of CFTR ARMS primers for multiplex reactions.
Initial denaturation was for 7.5 min at 95 °C followed by 40 cycles of 95 °C for 1 s, 64 °C for 20 s, and 72 °C for 20 s. A touchdown PCR was carried out with the annealing temperature starting at 70 °C and decreasing to 64 °C at 1 °C/cycle. Fluorescence was measured at the end of the annealing step. The PCR stage was followed by a melting curve analysis (95 °C for 10 s, 40 °C for 30 s, and heating back up to 70 °C at 0.1 °C/s) with continuous monitoring of fluorescence. The touchdown PCR conditions used allowed specific amplification of wild-type or mutated sequences as detected by standard agarose electrophoresis and melting curve analysis.
During the melting curve analysis, a temperature will be reached that causes the fluorescence resonance energy transfer to decrease as the detector probe is disassociated from its complementary sequence adjacent to the anchor probe. The first derivative of the melting curve is determined, which allows the decrease in fluorescence to be seen as a peak. When we used the MeltCalc software to assist in appropriate detector probe design, the actual Tms seen during a melting curve analysis were within 1–2 °C of the predicted Tms for the three detector probes. CF4ARMS-P, CF10ARMS-P, and CF11ARMS-P produced Tm peaks during melting curve analysis of 62, 51, and 55 °C, respectively. In the melting curve profile of a representative sample shown in Fig. 1 , the orientation of melting curve peaks can be clearly seen.
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Figure 1. Melting curve profile of a R117H/G551D compound heterozygote.
The F2 channel (top plot) detects both the wild-type and mutant F508del and R117H ARMS products with their respective peaks seen at 51 and 62 °C. The F3 channel (bottom plot) detects the G551D ARMS products with a peak seen at 55 °C. Reactions A and B are set up as given in Table 1 . The two peaks seen in the F2 channel at 62 °C and in the F3 channel at 55 °C show that this patient is a carrier for both mutations. A single peak at 51 °C in the F2 channel for the A reaction shows that only the wild-type F508del ARMS primers have produced a product for this sample. The agarose gel image also correlates with the melting curve peaks.
Using the optimized protocol as outlined in the methods, we blind-tested a panel of patient samples containing combinations of the five mutations. All patient samples were correctly identified and correlated with previous genotyping carried out by standard techniques.
The main advantage of the LightCycler system is its speed: the above protocols require 45 min. Genotyping on the LightCycler system with standard multiplex hybridization probes that overlie the mutation site can be technically challenging and have some limitations. Even with careful design of the hybridization probes, wild-type and mutant alleles may not necessarily be differentiated from each other (10). Our system allows hybridization probes to bind to any region of the amplicon, therefore providing scope for better probe design. The number of mutations that can be detected in a single reaction is dependent on the number of melting peaks that can be clearly differentiated from each other (11). This usually leads to development of multiplex reactions in which a single mutation is detected in a color channel. Here we have demonstrated a method that makes it quite possible to detect more than one mutation in a single channel. By use of the nearest-neighbor formula in the MeltCalc software, detector probes can be carefully designed that produce a peak within 1–2 °C of the predicted Tm. This allows Tm peaks, which indicate the presence or absence of a PCR product, to be strategically placed within the typical melting curve range of 45–70 °C for hybridization probes. Over this melting curve range we feel it should be possible to clearly distinguish three peaks in a single channel. This potentially makes it possible to multiplex six mutations in two capillaries when both fluorescent channels are used. This enhances the capabilities of the LightCycler for mutation detection; similar strategies could also be used for other real-time systems.
The results presented here demonstrate the ability to carry out multiplex mutation detection by use of a combination of ARMS PCR and real-time detection. A laboratory currently using ARMS PCR in a diagnostic setting can quite easily convert the standard ARMS PCR to a real-time ARMS PCR. This could have a major advantage in time savings and reduce the handling of potential carcinogenic ethidium bromide.
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Clinical Chemistry 50: 773-775, 2004; 10.1373/clinchem.2003.030445
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(Clinical Chemistry. 2004;50:773-775.)
© 2004 American Association for Clinical Chemistry, Inc.
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Technical Briefs
Detection of Five Common CFTR Mutations by Rapid-Cycle Real-Time Amplification Refractory Mutation System PCR
Eugene Dempsey1, David E. Barton2 and Fergus Ryan1,a
1 Department of Biological Sciences, Dublin Institute of Technology, Dublin, Ireland;2 National Centre for Medical Genetics and Department of Paediatrics, University College Dublin, Our Lady’s Hospital for Sick Children, Crumlin, Dublin, Ireland
aaddress correspondence to this author at: Department of Biological Sciences, Dublin Institute of Technology, Kevin St., Dublin 8, Ireland; e-mail fergus.x.ryan@dit.ie
Cystic fibrosis is the most common autosomal recessive disease in Caucasian populations and has a carrier frequency of 1 in 25 (1). The gene involved codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane-associated protein involved in ion transport across the plasma membrane of epithelial cells. To date more than 1000 mutations have been described in this gene, and most are rare (2). By focusing on five common mutations it is possible to detect the disease-causing mutation in 90% of Irish patients (3). The five mutations [and the percentages of Irish (3) and worldwide (2) cases] are F508del (77.4%, 66.0%), G551D (7.1%, 1.6%), R117H (2.7%, 0.3%), 621+1 G>T (1.4%, 0.7%), and G542X (0.5%, 2.4%). Four of these fall into the severe class of mutations in which the mRNA is incorrectly spliced (621+1 G>T), or in which the protein is not synthesized (G542X) or is blocked during processing (F508del), or its regulation is blocked (G551D).
Mutations in the CFTR gene can be detected by many mutation-detection systems, including single-strand conformation polymorphism analysis (4), restriction fragment length polymorphism analysis, Amplification Refractory Mutation System (ARMS) PCR (5), and more recently, real-time PCR systems using Sybr Green 1 (6) or hybridization probes (7). Currently, for mutation detection using hybridization probes on the LightCycler system, the detector probe is designed to overlie the possible site of the mutation. This can have drawbacks when multiplexing in the same detection channel because peaks can become merged, making genotyping of a sample difficult.
This study aimed to improve the multiplexing capabilities of real-time PCR in its use for mutation detection. To this end we used ARMS PCR primers to selectively amplify the wild-type or mutant alleles in separate reactions. Subsequent detection of PCR products was carried out with a common set of hybridization probes. The advantage of this system is that it increases flexibility in design of the hybridization probe, allowing probes with precise melting temperatures (Tms) to be generated. In this way melting curve peaks can be at a predetermined Tm for a particular PCR product, which allows for easier multiplexing of real-time PCR. Similar ARMS-based techniques have been developed for the ABI Prism 7700 sequence detector from PE Applied Biosystems using TaqMan® probes (8). These protocols depend on the detection of an amplification curve and are limited to the detection of one mutation per color channel. Our protocol detects melting curve peaks post amplification and allows multiple mutations to be detected per color channel.
Both control samples for protocol optimization and test samples for the blinded study were obtained from the National Centre for Medical Genetics, Our Lady’s Hospital for Sick Children (Crumlin, Dublin, Ireland). This study has received ethical approval from the DIT ethical review board (Project Ref. SF01/02).
Two multiplex PCR reactions were optimized by use of previously published ARMS primers (5). The first reaction detects the G551D, R117H, and F508del mutations (87.2% frequency in Ireland, 67.9% worldwide). The F508del ARMS primers (CF-DFjN and CF-DFwM) are specific for this mutation and are not influenced by the benign I506V and F508C mutations. Because the CF-DFwM primer is complementary to the sequence distal to F508del, the presence of the I507del mutation is not recognized (5).
The second reaction detects the 621+1 G>T and G542X mutations (1.9% frequency in Ireland, 3.1% worldwide). A PCR product should be formed in both the A and B ARMS reactions regardless of the sample genotype.
Three sets of fluorescence resonance energy transfer hybridization probes were designed to detect the 10 different ARMS PCR products. R117H and 621+1 G>T ARMS products are detectable by the CF4ARMS-A (5'-CCTTTTGTAGGAAGTCACCAAAGCAGTAC-F-3') and CF4ARMS-P (5'-LCRed640-GCCTCTCTTACTGGGAAGAATCA-P-3') hybridization probes (F indicates a fluorescein, and P indicates phosphate). The F508del ARMS products are detected by the CF10ARMS-A (5'-GCACAGTGGAAGAATTTCATTCTGTTCTCAG-F-3') and CF10ARMS-P (5'-LCRed640-TTCCTTGGATTATGCCT-P-3') hybridization probes. The CF11ARMS-A (5'-TATGATTACATTAGAAGGAAGATGTGCCTTT-F-3') and CF11ARMS-P (5'-LCRed705-AATTCAGATTGAGCATACT-P-3') hybridization probes detect both the G551D and G542X ARMS products. The predicted Tms of the detector probes were calculated by use of MeltCalc software (www.meltcalc.com) (9). All probes and primers were synthesized by Proligo, France.
ARMS PCR was carried out on a LightCycler (Roche Diagnostics). The total reaction volume was 10 µL, containing 1 µL of 10x LightCycler DNA Master Hybridization enzyme mixture (Roche Diagnostics), 3 mM MgCl2, 0.1 µM hybridization probes, and ARMS primers concentrations as outlined in Table 1 . ARMS PCR and subsequent melting curve analysis were carried out as follows:
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Table 1. Concentrations of CFTR ARMS primers for multiplex reactions.
Initial denaturation was for 7.5 min at 95 °C followed by 40 cycles of 95 °C for 1 s, 64 °C for 20 s, and 72 °C for 20 s. A touchdown PCR was carried out with the annealing temperature starting at 70 °C and decreasing to 64 °C at 1 °C/cycle. Fluorescence was measured at the end of the annealing step. The PCR stage was followed by a melting curve analysis (95 °C for 10 s, 40 °C for 30 s, and heating back up to 70 °C at 0.1 °C/s) with continuous monitoring of fluorescence. The touchdown PCR conditions used allowed specific amplification of wild-type or mutated sequences as detected by standard agarose electrophoresis and melting curve analysis.
During the melting curve analysis, a temperature will be reached that causes the fluorescence resonance energy transfer to decrease as the detector probe is disassociated from its complementary sequence adjacent to the anchor probe. The first derivative of the melting curve is determined, which allows the decrease in fluorescence to be seen as a peak. When we used the MeltCalc software to assist in appropriate detector probe design, the actual Tms seen during a melting curve analysis were within 1–2 °C of the predicted Tms for the three detector probes. CF4ARMS-P, CF10ARMS-P, and CF11ARMS-P produced Tm peaks during melting curve analysis of 62, 51, and 55 °C, respectively. In the melting curve profile of a representative sample shown in Fig. 1 , the orientation of melting curve peaks can be clearly seen.
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Figure 1. Melting curve profile of a R117H/G551D compound heterozygote.
The F2 channel (top plot) detects both the wild-type and mutant F508del and R117H ARMS products with their respective peaks seen at 51 and 62 °C. The F3 channel (bottom plot) detects the G551D ARMS products with a peak seen at 55 °C. Reactions A and B are set up as given in Table 1 . The two peaks seen in the F2 channel at 62 °C and in the F3 channel at 55 °C show that this patient is a carrier for both mutations. A single peak at 51 °C in the F2 channel for the A reaction shows that only the wild-type F508del ARMS primers have produced a product for this sample. The agarose gel image also correlates with the melting curve peaks.
Using the optimized protocol as outlined in the methods, we blind-tested a panel of patient samples containing combinations of the five mutations. All patient samples were correctly identified and correlated with previous genotyping carried out by standard techniques.
The main advantage of the LightCycler system is its speed: the above protocols require 45 min. Genotyping on the LightCycler system with standard multiplex hybridization probes that overlie the mutation site can be technically challenging and have some limitations. Even with careful design of the hybridization probes, wild-type and mutant alleles may not necessarily be differentiated from each other (10). Our system allows hybridization probes to bind to any region of the amplicon, therefore providing scope for better probe design. The number of mutations that can be detected in a single reaction is dependent on the number of melting peaks that can be clearly differentiated from each other (11). This usually leads to development of multiplex reactions in which a single mutation is detected in a color channel. Here we have demonstrated a method that makes it quite possible to detect more than one mutation in a single channel. By use of the nearest-neighbor formula in the MeltCalc software, detector probes can be carefully designed that produce a peak within 1–2 °C of the predicted Tm. This allows Tm peaks, which indicate the presence or absence of a PCR product, to be strategically placed within the typical melting curve range of 45–70 °C for hybridization probes. Over this melting curve range we feel it should be possible to clearly distinguish three peaks in a single channel. This potentially makes it possible to multiplex six mutations in two capillaries when both fluorescent channels are used. This enhances the capabilities of the LightCycler for mutation detection; similar strategies could also be used for other real-time systems.
The results presented here demonstrate the ability to carry out multiplex mutation detection by use of a combination of ARMS PCR and real-time detection. A laboratory currently using ARMS PCR in a diagnostic setting can quite easily convert the standard ARMS PCR to a real-time ARMS PCR. This could have a major advantage in time savings and reduce the handling of potential carcinogenic ethidium bromide.
Mutation detection using a novel plant endonuclease
CA Oleykowski, CR Bronson Mullins, AK Godwin and AT Yeung
Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA.
We have discovered a useful new reagent for mutation detection, a novel nuclease CEL I from celery. It is specific for DNA distortions and mismatches from pH 6 to 9. Incision is on the 3'-side of the mismatch site in one of the two DNA strands in a heteroduplex. CEL I-like nucleases are found in many plants. We report here that a simple method of enzyme mutation detection using CEL I can efficiently identify mutations and polymorphisms. To illustrate the efficacy of this approach, the exons of the BRCA1 gene were amplified by PCR using primers 5'-labeled with fluorescent dyes of two colors. The PCR products were annealed to form heteroduplexes and subjected to CEL I incision. In GeneScan analyses with a PE Applied Biosystems automated DNA sequencer, two independent incision events, one in each strand, produce truncated fragments of two colors that complement each other to confirm the position of the mismatch. CEL I can detect 100% of the sequence variants present, including deletions, insertions and missense alterations. Our results indicate that CEL I mutation detection is a highly sensitive method for detecting both polymorphisms and disease- causing mutations in DNA fragments as long as 1120 bp in length.
CA Oleykowski, CR Bronson Mullins, AK Godwin and AT Yeung
Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA.
We have discovered a useful new reagent for mutation detection, a novel nuclease CEL I from celery. It is specific for DNA distortions and mismatches from pH 6 to 9. Incision is on the 3'-side of the mismatch site in one of the two DNA strands in a heteroduplex. CEL I-like nucleases are found in many plants. We report here that a simple method of enzyme mutation detection using CEL I can efficiently identify mutations and polymorphisms. To illustrate the efficacy of this approach, the exons of the BRCA1 gene were amplified by PCR using primers 5'-labeled with fluorescent dyes of two colors. The PCR products were annealed to form heteroduplexes and subjected to CEL I incision. In GeneScan analyses with a PE Applied Biosystems automated DNA sequencer, two independent incision events, one in each strand, produce truncated fragments of two colors that complement each other to confirm the position of the mismatch. CEL I can detect 100% of the sequence variants present, including deletions, insertions and missense alterations. Our results indicate that CEL I mutation detection is a highly sensitive method for detecting both polymorphisms and disease- causing mutations in DNA fragments as long as 1120 bp in length.
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