Direct PCR on hair, saliva, cheek scrub and dead skin lysates
TransDirect® Animal Tissue Direct PCR Kit
Genotyping is the process of identifying differences in the genetic make-up (genotype) of an individual by examining the individual’s DNA sequence and comparing it to another specimen or a reference sequence. It reveals the alleles an individual has inherited from their parents. Traditionally, genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual. Genotyping can be performed on purified DNA material by PCR or on unpurified DNA by Direct PCR.
Taq DNA polymerase is a common enzyme used by researchers who use transgenic mice for their research. Mice genotyping is part of a weekly or monthly routine in many labs because it enables them select the correct mice to use in their experiments. For some high-throughput labs, it is essential to find a reliable method for rapid and efficient extraction of genomic DNA and PCR. One could use nucleic acid extraction kits to first extract and purify gDNA from their samples and then proceed to PCR with EasyTaq DNA polymerase or any other of Transgen Biotech’s high-performance DNA polymerases. However, imagine having to process hundreds of samples per week.
Various methods for extracting genomic DNA exist. The most popular involve using Proteinase K and genomic DNA extraction kits using silica-based spin columns. The cleanliness of the genomic DNA extract is a factor influencing DNA polymerase efficiency because it may contain molecules that inhibit DNA Polymerase activity. Nevertheless, commercial Taq formulations which can resist DNA polymerase inhibitors present in crude tissues or cell samples are now offered, allowing for direct PCR without prior DNA extraction.
The Transgen Biotech TransDirect® product line includes TransDirect® Animal Tissue PCR Kit. This kit uses a proprietary buffer in which samples are incubated for 10 min at room temperature and then heated 3 min at 95 °C. Lysates are then combined with 2x TransDirect PCR supermix containing a special inhibitor-resistant DNA polymerase (TransBD) and specific primers to perform PCR. The whole process requires much less time and ressources (no centrifugation), less pipetting steps than column-based gDNA extraction. This process is thus much more suitable for high-throughput labs.
Our goal was to demonstrate the efficiency of TransDirect® Animal Tissue kit on samples taken from…myself (Simon)! This demonstration is also used as a prelude to future product comparisons and performance tests which will be linked here once they are published on our website.
RESULTS & DISCUSSION
Scratching your nails won’t work but everything else will!
As depicted in Figure 1, the TransDirect Animal PCR kit successfully lysed cells from hair, saliva, cheek and dead skin but not from trying to cratch something off (my) nail. Direct PCR amplification of the 1-exon human gene MC2R using the provided supermix was successful with all lysates except nail scrub.
The procedure was fast, efficient and can be recommended for high-throughput labs, clinicians or small labs without not equipped with a 15 000 g centrifuge normally used with column-based extraction methods.
Future tests will assess wether standard and high-processivity DNA polymerases can perform well using the TransDirect lysates in Direct PCR, high-fidelity PCR and GC-rich PCR.
Figure 1. TransDirect Animal Tissue PCR Kit. Lysis and Direct PCR reactions were performed as described here. pcDNA5/FRT/Myc-MC2R and pcDNA5/FRT were used as positive and negative controls respectively.
Materials and Methods
Figure 2. The miniPCR system and BlueGel electrophoresis and transillumination system were obtained from Amplyus LLC (Cambridge, MA, USA). The BlueGel system uses LED blue-light illumination.
Agarose (GS201-01), ddH2O (GI101-01), 6x DNA loading dye (GH101-01), dNTPs (AD101) and 100bp DNA ladder (BM311) were obtained from our stocks at Civic Bioscience (www.civicbio.com; St-Mathieu-de-Beloeil, QC, Canada) and originate from Transgen Biotech (Beijing, China). GreenView Plus DNA stain was obtained from miniPCR – Amplyus.
Genomic DNA extraction with TransDirect Animal Tissue PCR Kit buffers
As instructed by the datasheet: TransDirect Animal Tissue PCR Kit. The hair sample was incubated at 55 °C for 10 min instead of at room temperature in the first step.
PCR reaction setup
25 ul reactions were setup on ice as follows:
- ddH2O up to 25 ul
- 12.5 ul TransDirect PCR mastrmix
- 2 uM forward primer
- 2 uM reverse primer
- 3 ul of lysate
1- Initial denaturation: 2 min at 94 °C
2- 35 cycles
- Denaturation: 94 °C for 30s
- Annealing: 53 °C for 15 s
- Extension: 72 °C for 45 s
3- Final extension : 72 °C for 120 s
Samples were mixed with DNA loading buffer and run on 1.5 % agarose gels in TAE buffer.