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PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine 5'-triphosphate.
Flores-Juárez, Cyntia R; González-Jasso, Eva; Antaramian, Anaid; Pless, Reynaldo C.
Affiliation
  • Flores-Juárez CR; CICATA, Instituto Politécnico Nacional, Querétaro, Querétaro, 76090, Mexico.
  • González-Jasso E; CICATA, Instituto Politécnico Nacional, Querétaro, Querétaro, 76090, Mexico.
  • Antaramian A; Instituto de Neurobiología, Universidad Nacional Autónoma de México, Juriquilla, Querétaro, 76230, Mexico.
  • Pless RC; CICATA, Instituto Politécnico Nacional, Querétaro, Querétaro, 76090, Mexico.
Biotechniques ; 61(4): 175-182, 2016 10 01.
Article in En | MEDLINE | ID: mdl-27712580
ABSTRACT
GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.
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Collection: 01-internacional Database: MEDLINE Main subject: DNA / Polymerase Chain Reaction / GC Rich Sequence / Deoxycytosine Nucleotides Limits: Humans Language: En Journal: Biotechniques Year: 2016 Document type: Article Affiliation country: Mexico
Search on Google
Collection: 01-internacional Database: MEDLINE Main subject: DNA / Polymerase Chain Reaction / GC Rich Sequence / Deoxycytosine Nucleotides Limits: Humans Language: En Journal: Biotechniques Year: 2016 Document type: Article Affiliation country: Mexico