Universal insertion/deletion-enrich PCR.
OBJECTIVE
Insertions and deletions (indels) are sometimes critical genetic events and can lead to various diseases. Identifying indel types by DNA sequencing is difficult and labor intensive. The sensitivity of DNA sequencing techniques such as Sanger's direct sequencing, which broadly screens all types of microindels in predefined areas, is somewhat limited. Other techniques such as indel variant-specific primer sets are sensitive and specific, but are expensive, labor intensive, less efficient and only cover known indel variants.


METHODS
We report on the universal insertion/deletion-enrich PCR (Unindel-PCR) which has ultra-high sensitivity and can be used to screen for known and unknown indels in a predefined area. In brief, the Unindel-PCR consists of one forward, one reverse and one blocking primer. At very condition, blocking primer is superior to forward primer in recognizing the wild-type template and prevent nucleotide extension and wild-type amplification during PCR cycles. Conversely, forward primer is superior to blocking primer in recognizing the indel template and promote indel-type amplification during PCR cycles.


RESULTS
Unindel-PCR performs well in detecting any indels in a wild-type background. The sensitivity is 1% in Unindel-PCR and 0.01% in nested Unindel-PCR. The wild-type inhibition capacity of Unindel-PCR is 10(7). One thousand copies of deletion type plasmids are sufficient for detection by Unindel-PCR, whereas nested Unindel-PCR requires only 10 copies.


CONCLUSIONS
Unidel-PCR will remold the capability of PCR-based genetic testing, especially in the field of cancer molecular diagnosis, infectious disease and identification of minor populations of alternative splicing variants of RNA transcribed.
Department of Pathology, Mackay Memorial Hospital, Taipei, Taiwan. chenchikuan@googlemail.com
Chi-Kuan
CK
Chen
Jr-Kai
JK
Huang
D005091:Exons; D018773:Genes, erbB-1; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D054643:INDEL Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity