Simplified hot start PCR

Birch, David; Kolmodin, Lori A.; Wong, Jasmine; Zangenberg, G.; Zoccoli, Michael; McKinney, Nancy; Young, Katherine

doi:10.1038/381445a0

Cited by 140 publications

(56 citation statements)

References 4 publications

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“…To further minimize the formation of the template-independent, artifactual product primer-dimer (Chou et al 1992), we used a modified, "Gold" version of the Stoffel fragment polymerase (Birch 1996) to provide a simplified hot start. Additionally, enough ROX dye was added to the PCR reactions to provide a significant level of fluorescence at "baseline" reads.…”

Section: Reaction Optimization and Pcr Amplificationmentioning

confidence: 99%

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

Germer

Holland²,

Higuchi³

2000

Genome Res.

396

295

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We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r 2 = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.

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Section: Reaction Optimization and Pcr Amplificationmentioning

confidence: 99%

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

Germer

Holland²,

Higuchi³

2000

Genome Res.

396

295

View full text Add to dashboard Cite

show abstract

“…Such a "hot-start" has been made most convenient through the use of a chemically-modified DNA polymerase (AmpliTaq Gold TM , Perkin-Elmer, Norwalk, CT; Birch, 1996) that requires an incubation at greater than 90 deg C. to be activated. This allows the enzyme to be present during PCR setup.…”

Section: Enhancing Specificity and Sensitivitymentioning

confidence: 99%

Kinetic PCR analysis using a CCD camera and without using oligonucleotide probes

Higuchi

Watson

1999

PCR Applications

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“…12,13 Although the optimum temperature for Taq DNA polymerase is between 60°C and 72°C, it will work at room temperature, although relatively slowly. This can lead to poor amplification, and potentially amplification of the wrong fragments if there is a time delay between preparing the PCR and adding it to the thermal cycler.…”

Section: Amplification Of Str Loci By Pcrmentioning

confidence: 99%

Forensic DNA profiling: state of the art

Linacre¹,

Templeton²

2014

RRFMS

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DNA profiling is now a routine test, yet it has only been 26 years since its first use. During these intervening years, the science of DNA profiling has undergone many changes, and it will continue to do so in the future. DNA profiling in the laboratory starts with DNA extractions and is followed by quantification of the DNA, amplification of up to 23 human specific short tandem repeat loci, and separation on capillary columns. The final stage is interpretation of the data leading to reporting the DNA evidence to a court. Each of these steps has seen improvements leading to higher sensitivity from minute trace samples. Genetic markers on the X and Y chromosomes are now used for both criminal and civil investigations, along with mitochondrial DNA. Mitochondrial DNA has been used in forensic science since the 1990s because of its high copy number and particular maternal inheritance. These sex chromosome markers and mitochondrial DNA were used effectively to determine the identity or ancestry of individuals, and now other genetic markers can be used to determine certain phenotypic traits to a high degree of accuracy. Genetic testing adapted from medical and pharmaceutical sciences, such as next-generation DNA sequencing, will soon be applied to mainstream forensic science, opening new avenues in criminal investigations. This review aims to cover these key events and advancements in the field from both a historical view and current practice. Keywords: forensic science, DNA profiling, short tandem repeats, mitochondrial DNA, phenotypic testing, next-generation sequencing Opening commentsDNA profiling has revolutionized the process of forensic human identification. It is of note that this is a relatively recent addition to the forensic science tool kit and that it is constantly undergoing developments. The aim of this review is to take the reader through the processes of DNA typing, starting with DNA isolation from biological material through novel approaches and phenotypic testing. The review is written for those in forensic medicine; therefore, certain knowledge is assumed, but equally, aspects that are specific to forensic science will be emphasized and placed into the right context. For those who wish more information than can be provided within the scope of this review, there are books ranging from introductory texts 1 to those that are more comprehensive.2,3

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Simplified hot start PCR

Cited by 140 publications

References 4 publications

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

Kinetic PCR analysis using a CCD camera and without using oligonucleotide probes

Forensic DNA profiling: state of the art

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