Abstract

The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors. An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n·(dG-dT)n micro-satellites in the polymerase chain reaction (PCR). These products differ from the primary product by multiples of 2 nucleotides. We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n·(dG-dT)n frameshifting during the PCR. Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification. However, 3′→5′ exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3′ terminal nucleotides. Most interestingly, extension temperatures of 37°C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3′→5′ exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products. This method can improve the resolution of heterozygous or mutant (dC-dA)n·(dG-dT)n alleles differing in size by one or two repeat units.

Original languageEnglish (US)
Pages (from-to)2429-2434
Number of pages6
JournalNucleic acids research
Volume24
Issue number12
DOIs
StatePublished - Jun 15 1996

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Dilatation and Curettage
DNA-Directed DNA Polymerase
Microsatellite Repeats
Exonucleases
Polymerase Chain Reaction
DNA
Nucleotides
DNA Polymerase I
Sequence Deletion
Genomic Instability
Insertional Mutagenesis
Alleles
Temperature
Neoplasms
bacteriophage T7 induced DNA polymerase

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

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title = "Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n·d(G-T)n microsatellite repeats",
abstract = "The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors. An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n·(dG-dT)n micro-satellites in the polymerase chain reaction (PCR). These products differ from the primary product by multiples of 2 nucleotides. We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n·(dG-dT)n frameshifting during the PCR. Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification. However, 3′→5′ exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3′ terminal nucleotides. Most interestingly, extension temperatures of 37°C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3′→5′ exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products. This method can improve the resolution of heterozygous or mutant (dC-dA)n·(dG-dT)n alleles differing in size by one or two repeat units.",
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Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n·d(G-T)n microsatellite repeats. / Hite, Jill M.; Eckert, Kristin; Cheng, Keith.

In: Nucleic acids research, Vol. 24, No. 12, 15.06.1996, p. 2429-2434.

Research output: Contribution to journalArticle

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T1 - Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n·d(G-T)n microsatellite repeats

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AU - Eckert, Kristin

AU - Cheng, Keith

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N2 - The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors. An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n·(dG-dT)n micro-satellites in the polymerase chain reaction (PCR). These products differ from the primary product by multiples of 2 nucleotides. We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n·(dG-dT)n frameshifting during the PCR. Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification. However, 3′→5′ exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3′ terminal nucleotides. Most interestingly, extension temperatures of 37°C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3′→5′ exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products. This method can improve the resolution of heterozygous or mutant (dC-dA)n·(dG-dT)n alleles differing in size by one or two repeat units.

AB - The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors. An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n·(dG-dT)n micro-satellites in the polymerase chain reaction (PCR). These products differ from the primary product by multiples of 2 nucleotides. We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n·(dG-dT)n frameshifting during the PCR. Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification. However, 3′→5′ exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3′ terminal nucleotides. Most interestingly, extension temperatures of 37°C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3′→5′ exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products. This method can improve the resolution of heterozygous or mutant (dC-dA)n·(dG-dT)n alleles differing in size by one or two repeat units.

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