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Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
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Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp
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Nested Patch PCR for highly multiplexed amplification of genomic loci.

Katherine E Varley1, Robi D Mitra

  • 1Department of Genetics, Center for Genome Sciences, Washington University School of Medicine, St. Louis, MO 63108, USA.

Cold Spring Harbor Protocols
|February 12, 2010
PubMed
Summary

Nested Patch polymerase chain reaction (PCR) amplifies over 90 DNA loci simultaneously for mutation detection. This method enables parallel SNP and mutation identification across numerous samples and loci efficiently.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Accurate detection of single nucleotide polymorphisms (SNPs) and mutations is crucial for genetic research and diagnostics.
  • Existing methods for large-scale targeted sequencing can be complex and time-consuming.

Purpose of the Study:

  • To introduce and validate Nested Patch polymerase chain reaction (PCR) as an efficient method for simultaneous amplification of numerous targeted DNA loci.
  • To demonstrate the utility of Nested Patch PCR for high-throughput SNP and mutation detection.

Main Methods:

  • Simultaneous amplification of >90 targeted loci from genomic DNA using Nested Patch PCR.
  • Sequencing of amplified loci using second-generation sequencing platforms.
  • Application of sample-specific DNA barcodes for multiplexing and pooling samples.

Main Results:

  • Nested Patch PCR achieved high specificity, with 90% of sequencing reads mapping to targeted loci.
  • The protocol allows for parallel processing of multiple samples through pooling and barcoding.
  • Successful identification of SNPs and mutations across many targeted loci in parallel.

Conclusions:

  • Nested Patch PCR offers a streamlined workflow for identifying SNPs and mutations across numerous targeted loci.
  • The method is suitable for high-throughput genetic analysis involving multiple samples.
  • This technique facilitates efficient genetic variant discovery in large-scale studies.