Applications

LoTemp™ HiFi^® ready-to-use PCR mix has been successfully used for general DNA Amplification, reverse transcription-PCR and realtime PCR.

Genetically modified Bacillus stearothermophilus (Bst) DNA polymerases are highly processive enzymes, capable of repeated nucleotide primer extension under a wide range of matrix conditions, even in the presence of competing non-target nucleic acids and in the presence of DNA melting agents which are incorporated in the matrix to lower the denaturing temperature during PCR thermocycling.

One of the applications of HiFi^® DNA polymerases in LoTemp™ PCR is to amplify the hypervariable region of the L1 gene of human papillomavirus (HPV) released from cervicovaginal cells for accurate genotyping by direct DNA sequencing.

The species-specific opacity protein (opa) genes of Neisseria gonorrhoeae and the species-specific cryptic plasmid DNA of Chlamydia trachomatis in the cervicovaginal specimens can be also amplified with appropriate primers and validated by direct DNA sequencing.

With the HiFi^® DNA polymerase LoTemp™ PCR system, proteinase K digestates of alcohol-fixed cervicovaginal cell samples can be used as templates for target DNA amplification without purification. The nested PCR products are suitable for direct cycle sequencing.

For more information, please read the article published in Infectious Agents and Cancer and American Journal of Clinical Pathology visit the website www.hpvtyping.com provided by one of our regular customers. Examples of the PCR products and signature DNA sequencing are illustrated as follows.

Figure 1. Selected PCR amplicons generated by primer pairs

Lane 1= ATCC HPV-16 L1 genomic DNA primary outer PCR amplicon ~ 450 bp.
Lane 2= ATCC HPV-16 L1 genomic DNA nested PCR amplicon ~190 bp.
Lane 3= HPV-positive cervicovaginal sample with an L1 primary outer PCR amplicon ~ 450 bp.
Lane 4= HPV-positive cervicovaginal sample with an L1 nested PCR amplicon ~ 190 bp.

Lane 5= ATCC 49226 N. gonorrhoeae opa gene primary outer PCR amplicons ~ 210-270 bp.
Lane 6= ATCC 49226 N. gonorrhoeae opa gene nested PCR amplicons ~ 127-130 bp.
Lane 7= Cervicovaginal sample with gonococcal opa gene primary outer PCR amplicons ~ 210-270 bp.
Lane 8= Cervicovaginal sample with gonococcal opa gene nested PCR amplicons ~ 127-130 bp.

Lane 9= ATCC VR-886 C. trachomatis cryptic plasmid DNA primary outer PCR amplicon ~ 290 bp.
Lane 10= ATCC VR-886 C. trachomatis cryptic plasmid DNA nested PCR amplicon ~ 160 bp.
Lane 11= Cervicovaginal sample with a faint ~290 bp C. trachomatis cryptic plasmid DNA outer PCR amplicon.
Lane 12= Cervicovaginal sample with C. trachomatis cryptic plasmid nested PCR amplicon ~160 bp.

M = Molecular ruler 100-1000 bp in size.

NOTE: PCR, DNA sequencing technology and the reagents supplied by HiFi DNA Tech are for research and to be used by high complexity CLIA certified clinical laboratories only. Milford Medical Laboratory is one of such laboratories currently offering DNA sequencing services for validating HPV genotyping and for confirmation of N. gonorrhoeae opa gene and C. trachomatis cryptic plasmid DNA detected by PCR.