Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR.
Locked nucleic acid (LNA) is a modified DNA with increased binding affinityfor complementary DNA sequences. Our strategy was to use this property of LNA to inhibit undesired PCR amplification (e.g.,from contaminating genomic DNA) in a cDNA-based assay. By placing a short complementary LNA sequence in intronic DNA, the aim was to inhibit the amplification of genomic DNA without affecting the amplification of reverse-transcribed spliced mRNA. LNA was designed to bind within intron 5 in the x-box binding protein 1 (XBP1) gene. An irrelevant LNA oligonucleotide served as a negative control. In both PCR and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More than three DNA nucleotides reduced the LNA inhibition ability. The sequence specificity of the LNA was tested by investigating the number of LNA nucleotide mismatches permitted. The introduction of one mismatch maintained the inhibition of genomic amplification whereas two mismatches reduced the amplification. Our results show that LNA may be used to enhance the specificity of PCR by eliminating unwanted PCR products.
Hummelshoj
Lone
L
National University Hospital, Laboratory of Medical Allergology, Copenhagen, Denmark. l.hummelshoj@rh.dk
Ryder
Lars P
LP
Madsen
Hans O
HO
Poulsen
Lars K
LK
D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005784:Gene Amplification; D007438:Introns; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism; D009696:Nucleic Acids / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D000071701:Regulatory Factor X Transcription Factors; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013696:Temperature; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009696:Nucleic Acids; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D004247:DNA