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PCR Samples and Template DNA

Template DNA and Samples for PCR

Guidelines for template DNA and samples for PCR:

PCR Samples:  Types, Amount and Purity

Types of Samples for PCR

Single- or double-stranded DNA of any origin may be a PCR sample. Templates that can be used for amplification include animal, bacterial, plant, or viral. Conversion of RNA molecules, including total RNA, poly (A+) RNA, viral RNA, tRNA, or rRNA must occur prior to amplification when using these as templates to so-called complementary DNA (cDNA) by the enzyme reverse transcriptase (either MuLV or recombinant, rTth DNA polymerase).

Amount of Material for PCR

Starting material amount, required for PCR can be as little as a single molecule.  As a basis, up to nanogram amounts of DNA cloned template, up to microgram amounts of genomic DNA, or up to 105 DNA target molecules are best for initial PCR testing.

Purity of Template and Sample for PCR

Purity of the DNA sample that will be used for PCR amplification does not need not be high. A single cell, a crude cell lysate, or even a small sample of degraded DNA template is usually adequate for successful amplification. The requirements of sample purity must be that the target contains at least one intact DNA strand encompassing the amplified region and that the impurities associated with the target be dilute so as to not inhibit enzyme activity. Be that as it may, for some amplifications, such as long PCR, it may be necessary to consider the quality and quantity of the DNA sample. For example: 1. When more template molecules are available, there is less occurrences of false positives caused by either cross-contamination between samples or “carryover” contamination from previous PCR amplifications.

2. When the PCR amplifications lacks specificity or efficiency, or when the target sequences are limited, there is a greater chance of inadequate product yield.

3. When the fraction of starting DNA available to PCR is uncertain, it is increasingly difficult to determine the target DNA content.