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Abstract: We describe a method for rapidly amplifying whole genomes via a Phi29 DNA polymerase‐mediated strand displacement reaction (SDR). Genomic amplification products derived from the SDR reaction resulted in high quantities of DNA suitable for polymerase chain reaction (PCR) amplification and sequencing of mitochondrial genomes. Control region sequences of DNA derived directly from PCR amplicons of extracted DNA were identical to those derived from PCR amplification of SDR genomic DNA. Effective SDR amplification and subsequent sequencing was successful across tissues sources ranging in age from 1 year to 19 years. Strand replacement reaction genomic amplification offers a means of obtaining large quantities of DNA from small amounts of tissue.Keywords:
Multiple displacement amplification
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Multiple displacement amplification
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Multiple displacement amplification
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Abstract The polymerase chain reaction (PCR) is a temperature‐controlled technique for amplifying DNA in vitro . It requires a DNA template; two oligodeoxynucleotide primers, which define the DNA to be amplified; the four deoxynucleoside triphosphates; and a thermostable DNA polymerase. PCR is carried out in a thermocycler, in which the DNA is subjected to cycles of denaturation of the double‐stranded DNA template; annealing of the two primers, which are complementary to the two strands of the DNA template; and DNA polymerization (primer extension). Because the product of one cycle serves as the template for the next cycle, PCR leads to the exponential amplification of the initial DNA template, which produces more than a million copies of a homogenous PCR product in 20 cycles. PCR has become an indispensable technique in all life sciences and is used extensively for research and diagnostic purposes. PCR applications include cloning of genomic DNA, preparing DNA for sequencing, site‐directed mutagenesis and recombination, genetic fingerprinting for forensic purposes, detection and identification of infectious agents, prenatal diagnosis of genetic diseases, identification of allelic sequence variations, and gene‐expression analysis.
Hot start PCR
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The polymerase chain reaction (PCR)
1
was developed by Kerry Mullis in 1983 and is a mainstay of molecular biology. The history of the development of the PCR has been described
2
as well as its importance for biotechnology.
3
,
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PCR is a method used for amplification of DNA or RNA for analysis or use in recombinant DNA technology.
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–
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There are a number of version of the PCR which are shown in Table PCR 1. Some variations of the PCR such as the panhandle PCR
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,
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and vectorette PCR
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–
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which are used for genomic sequencing are not shown in the table. Although the bulk of PCR is used for genomic diagnostics and recombinant DNA work, there is use of PCR for the amplification of barcodes in DNA chemical libraries.
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,
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Primed in situ labeling (PRINS) is a technical approach related to PCR where a DNA probe is used to bind to denatured cellular DNA and serve as a primer for a PCR where the product can be visualized with a label such as biotin or digoxigenin (both as dUTP derivatives).
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,
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Protocols for the sequence analysis of conventional single-stranded or double-stranded DNA templates are often unsuitable for the direct sequencing of DNA fragments generated by the polymerase chain reaction (PCR) (1,2). The features that can distinguish PCR products as templates for sequencing include (a) contamination of the reactions by nonspecific PCR amplification products that are complementary to the sequencing primer, (b) the persistence of "leftover" PCR primers from the amplification reactions, and (c) the potential for competition between one strand of the amplified fragment and the oligonucleotide used for the sequencing. The various approaches that have been used to overcome these problems include
Multiple displacement amplification
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Abstract: We describe a method for rapidly amplifying whole genomes via a Phi29 DNA polymerase‐mediated strand displacement reaction (SDR). Genomic amplification products derived from the SDR reaction resulted in high quantities of DNA suitable for polymerase chain reaction (PCR) amplification and sequencing of mitochondrial genomes. Control region sequences of DNA derived directly from PCR amplicons of extracted DNA were identical to those derived from PCR amplification of SDR genomic DNA. Effective SDR amplification and subsequent sequencing was successful across tissues sources ranging in age from 1 year to 19 years. Strand replacement reaction genomic amplification offers a means of obtaining large quantities of DNA from small amounts of tissue.
Multiple displacement amplification
Amplicon
Applications of PCR
genomic DNA
In silico PCR
DNA nanoball sequencing
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Multiple displacement amplification
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Primer dimer
In silico PCR
Primer (cosmetics)
In vitro recombination
DNA nanoball sequencing
Hot start PCR
genomic DNA
Sequencing by ligation
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We present data on efficient amplification of large number of DNA targets using a single-tube polymerase chain reaction (PCR). This is a further enhancement of our approach to multiplexed PCR based on PCR suppression, which allows multiple DNA amplification using only one sequence-specific primer per amplicon while the second primer is common for all targets (Broude, N.E., et al., Proc. Natl. Acad. Sci. USA 98, 206-211, 2001). The reaction conditions have been optimized for simultaneous synthesis of 30 DNA targets, mostly consisting of fragments containing single nucleotide polymorphisms (SNP). The size of the amplified fragments, derived from many different human chromosomes, varies from 100 to 600 bp. We conclude that this method has potential for highly multiplexed DNA amplification useful for SNP analyses, DNA diagnostics, and forensics.
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Multiple displacement amplification
genomic DNA
Applications of PCR
Hot start PCR
Sequencing by ligation
DNA nanoball sequencing
Primer dimer
In silico PCR
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Amplicon
Applications of PCR
In silico PCR
Primer dimer
Hot start PCR
Multiple displacement amplification
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