AmpliTaq DNA polymerase, FS dye-terminator sequencing: analysis of peak height patterns - PubMed (original) (raw)
AmpliTaq DNA polymerase, FS dye-terminator sequencing: analysis of peak height patterns
L T Parker et al. Biotechniques. 1996 Oct.
Free article
Abstract
Taq DNA polymerases in which the phenylalanine is substituted by a tyrosine at position 667 (Taq F667Y) are members of a new class of DNA polymerases that incorporate chain-terminating dideoxyribonucleoside triphosphates (ddNTPs) much more efficiently than the wild-type Taq DNA polymerase. Improved incorporation of ddNTPs into DNA during cycle sequencing using AmpliTaq DNA polymerase, FS (Taq-FS, a member of the Taq F667Y family), and dye-labeled primers results in nearly uniform peak heights in the sequencing trace. This is not the case when dye-labeled ddNTPs are used in Taq-FS cycle sequencing reactions. While the rate of dye-terminator incorporation is more efficient with Taq-FS, the peak pattern is still highly variable and different from that produced by the wild-type enzyme. We have systematically examined pairs of sequence-tagged sites that vary at only a single nucleotide to determine how base changes influence the peak heights of neighboring bases in sequencing traces generated by the Taq-FS dye-terminator chemistry. In 31 of 64 possible 3-base windows (48%), we find that the peak height of a particular base can be predicted by knowing just one or two bases 5' to the base in question. We have also compared and contrasted the peak patterns produced by the Taq-FS enzyme with those previously identified for the wild-type enzyme. Establishing the patterns in peak heights within local sequence contexts can improve the accuracy of base-calling and the identification of polymorphisms/mutations when using the Taq-FS dye-terminator cycle-sequencing chemistry.
Similar articles
- Peak height variations in automated sequencing of PCR products using Taq dye-terminator chemistry.
Parker LT, Deng Q, Zakeri H, Carlson C, Nickerson DA, Kwok PY. Parker LT, et al. Biotechniques. 1995 Jul;19(1):116-21. Biotechniques. 1995. PMID: 7669285 - Peak height pattern in dichloro-rhodamine and energy transfer dye terminator sequencing.
Zakeri H, Amparo G, Chen SM, Spurgeon S, Kwok PY. Zakeri H, et al. Biotechniques. 1998 Sep;25(3):406-10, 412-4. doi: 10.2144/98253st01. Biotechniques. 1998. PMID: 9762437 - Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases.
Gardner AF, Jack WE. Gardner AF, et al. Nucleic Acids Res. 2002 Jan 15;30(2):605-13. doi: 10.1093/nar/30.2.605. Nucleic Acids Res. 2002. PMID: 11788725 Free PMC article. - PCR and DNA sequencing.
Gyllensten UB. Gyllensten UB. Biotechniques. 1989 Jul-Aug;7(7):700-8. Biotechniques. 1989. PMID: 2698653 Review. - Cycle sequencing.
Kretz K, Callen W, Hedden V. Kretz K, et al. PCR Methods Appl. 1994 Apr;3(5):S107-12. doi: 10.1101/gr.3.5.s107. PCR Methods Appl. 1994. PMID: 8038691 Review. No abstract available.
Cited by
- Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing.
Seroussi E. Seroussi E. Genes (Basel). 2021 Feb 17;12(2):283. doi: 10.3390/genes12020283. Genes (Basel). 2021. PMID: 33671263 Free PMC article. - Identification of Pneumocystis carinii f. sp. hominis gene sequences in filtered air in hospital environments.
Olsson M, Lidman C, Latouche S, Björkman A, Roux P, Linder E, Wahlgren M. Olsson M, et al. J Clin Microbiol. 1998 Jun;36(6):1737-40. doi: 10.1128/JCM.36.6.1737-1740.1998. J Clin Microbiol. 1998. PMID: 9620410 Free PMC article. - Application of sequence-specific labeled 16S rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization.
Rudi K, Skulberg OM, Skulberg R, Jakobsen KS. Rudi K, et al. Appl Environ Microbiol. 2000 Sep;66(9):4004-11. doi: 10.1128/AEM.66.9.4004-4011.2000. Appl Environ Microbiol. 2000. PMID: 10966421 Free PMC article. - Template-directed dye-terminator incorporation (TDI) assay: a homogeneous DNA diagnostic method based on fluorescence resonance energy transfer.
Chen X, Kwok PY. Chen X, et al. Nucleic Acids Res. 1997 Jan 15;25(2):347-53. doi: 10.1093/nar/25.2.347. Nucleic Acids Res. 1997. PMID: 9016564 Free PMC article. - A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome.
Vallone PM, Just RS, Coble MD, Butler JM, Parsons TJ. Vallone PM, et al. Int J Legal Med. 2004 Jun;118(3):147-57. doi: 10.1007/s00414-004-0428-5. Epub 2004 Feb 4. Int J Legal Med. 2004. PMID: 14760491
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources