The PCR (template) DNA must be a highly purified DNA having 30ng to 50ng concentration, 50% to 55% GC content and free from chemical contaminants and other DNA contaminants.
Inappropriate concentration fo the template DNA in PCR reaction results into reaction failure, likewise, a higher concentration of the DNA template in PCR, resulting in false positive results.
The Taq DNA polymerase can only be activated if it recognises the template DNA as a substrate for the PCR reaction. Although the polymerization cannot be started until the primer binds to the template DNA.
Similarly, the dTNPs cannot be binds to the site of amplification until the Taq recognised the PCR template DNA as a substrate for the reaction.
Therefore, the DNA used as a template in the PCR reaction plays an important role in amplification.
In this article, we will discuss several properties of the PCR DNA along with its concentration required to perform the PCR reaction.
The template DNA used in the PCR is isolated using several different types of DNA extraction methods. These DNA extraction methods are enlisted below,
Which types of DNA extraction method is selected for our protocol is depends on the types of tissue we are using,
For example, the phenol-chloroform DNA extraction method is a good choice for extracting DNA from the blood. similarly, the CTAB DNA extraction method is the best choice for plant tissue.
After the DNA extraction and before the polymerase chain reaction, the PCR (template) DNA must be quantified because the success of any PCR reaction depends on the quantity and the purity of the template DNA.
For quantification of the DNA, we can use two methods spectrophotometric method and fluorometric method. However, the spectroscopic method can do both quantification and purity check.
The DNA used for the PCR can be broadly divided into two categories:
- Target sequence DNA (the amount of DNA to be amplified)
- Non- target sequence DNA.
The target DNA is generally a smaller portion of the template DNA which is selected by us for the amplification, this may be a short or long sequence or an entire gene.
In general PCR reaction, the target DNA length is nearly 100bp to 1000bp or maximum 1500bp. For longer PCR which takes more time and required advance reaction preparation, the target DNA sequence may be 2000bp to 10,000bp long.
However, for the amplification of longer DNA fragment, more advanced protocol and other PCR additives are required.
The target sequence is < 1% of the total DNA.
The rest of the DNA is non-target sequence DNA which cannot be amplified into the reaction or it is a kind of junk DNA.
However, the non-target DNA can also hinder into the reaction.
Properties of PCR template DNA
The template DNA must be contaminant-free:
The genomic DNA used in the PCR reaction must be contaminant free. Phenol, CTAB, other inorganic and organic salts are the common contaminant of the DNA.
Proper washing and DNA purification are required before going to PCR.
If the purity of the DNA is above or below 1.8 (260/280 ratio), the PCR template DNA must be contaminated with proteins, phenol or RNA.
In that case, purify DNA before doing PCR otherwise the PCR amplification may not be achieved.
The template DNA must be free from other DNA contaminants:
PCR template DNA contaminated with the DNA of other organisms results in the false positive amplification into the PCR reaction.
For example, if we are doing tuberculosis PCR, there is a chance that along with the pathogen DNA the genomic DNA of the host might be amplified.
In this case, the primers bind to the non-complementary or semi-complementary sequences of other DNA and amplify it.
False positive results are one of the biggest problems in the PCR amplification. To avoid it purify the DNA before PCR.
GC content of the DNA:
The DNA is made up of the Adenine, Thymine, Cytosine and Guanine. Three hydrogen bonds between G and C and two hydrogen bonds between A and T joins two strands of DNA.
Due to more hydrogen bonds between G and C, the amplification of GC rich DNA is difficult. To avoid this, select the region of target DNA with GC content between 50% to 55%.
Higher GC content increases the melting temperature of the reaction, also it is very difficult to amplify it.
Concentration of template DNA:
30ng to 50ng DNA is required for 25 microliter PCR reaction. if the concentration is lower, the DNA might not be amplified. However, amplification is reported even with the 10pg of DNA.
Furthermore, a higher concentration of DNA also hinders in the PCR reaction. There are two possibilities for that, the DNA may amplify (non-specific) or the DNA may not be amplified.
Due to the higher concentration of DNA, the PCR tube is filled with a higher amount of non-specific DNA and hinders in the activity of the other reagents or the PCR reagents cannot be reached at the target DNA sequence which leads non-amplification and reaction failure.
In another possibility, due to the higher amount of the non-target template DNA, the primer bind to other than its complementary sequences and results in false positive PCR amplification.
Purity of template DNA:
Another important property of PCR (template) DNA is the purity of the DNA. The unpurified- contaminated DNA cannot be amplified.
Contaminants such as phenol, chloroform and other salt hinder in the PCR reaction. Phenol is one of common PCR inhibitor.
Tips: Always dissolve DNA in TE buffer and the pH of the TE buffer must be nearly 8.0. The integrity of the DNA maintained at the pH ~8.0.
Also, check the templet DNA if it is fragmented or not, fragmented DNA cannot be used in the PCR. For checking that use agarose gel electrophoresis technique.
If the concentration of the extracted DNA is too high, dilute it by using the TE buffer. Prepare a working stock of 30ng DNA for amplification.
Amplification is not possible without the PCR DNA template. why are saying it as PCR DNA template? because what DNA we are prepared (a purified 30ng DNA) can only be used for the PCR amplification. For other genomic techniques, the concentration of the total DNA may vary.