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Design considerations and effects of LNA in PCR primers.

Abstract：

:The effects of comprehensive LNA substitution in PCR primers for amplification of human genomic DNA targets are presented in this report. Previous research with LNA in other applications has shown interesting properties for molecular hybridization including enhanced specificity in allele-specific PCR. Here we systematically modified PCR primers and conditions for the human genomic DNA targets APOB and PAH, along with a beta-globin amplification control, to study whether the number and position of LNA residues improves or diminishes amplification sensitivity and specificity. It was observed that the design rules for LNA substitution in PCR primers are complex and depend upon number, position and sequence context. Technical advantages were seen when compared to DNA controls for the best LNA primer designs, which were typically one to a few centrally located LNA residues. LNA advantages include increased maximum annealing temperature (Tmax) and increased signal with limiting primer or Taq DNA polymerase. Several well-characterized designs exhibited different efficiencies with different brands of hot-start enzymes. Many shorter LNA primers were found to be functional compared to same-length non-functional native DNA controls. These results show that LNA-substituted PCR primers have potential for use in difficult PCR techniques, such as multiplex amplification at higher Tmax, once firm LNA primer design rules are established.

journal_name

Mol Cell Probes

journal_title

Molecular and cellular probes

authors

Latorra D,Arar K,Hurley JM

doi

10.1016/s0890-8508(03)00062-8

subject

Has Abstract

pub_date

2003-10-01 00:00:00

pages

253-9

issue

5

eissn

0890-8508

issn

1096-1194

pii

S0890850803000628

journal_volume

17

pub_type

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