The polymerase chain reaction(PCR) is known to be a very sensitive method of nucleic acid synthesis by which a particular segment of deoxyribonucleic acid(DNA) can be specifically amplified. However, as no single protocol will be appropriate to
all
situations, it requires to optimize the optimize the PCR conditions for a given application, especially analytical procedure to amplified fragment length polymorphism of VNTR region. Recently, typing of VNTR regions in a number of potential
problems
including generation of recombinant alleles during the extension phase.
The objective of this paper is to evaluate the cause of PCR products of abnormal length, and to set up the guideline for the reliable production of DIS80VNTR region in the human genome by studying several parameters that influence polymerase
chain
reaction.
In the study of DIS80 VNTR region to be amplified, the following PCR conditions are optimal in the 50 μl of reaction volume and under the temperature conditions of 95℃for 1 minute, 65℃for 2 minutes and 72℃for 2 minutes.
1. The adequate concentration of MgCI2 is within the range of 1.0 to 2.0mM.
2. The high activities of Taq polymerase show about 0.5 to 1.5 units without generation of the undesired products.
3. The adequate amount of template DNA is about 30 to 50ng, although the template DNA can be amplified with the minimum of 40pg.
4. With the 50ng of template DNA, the adequate numbers of PCR cycle range 25 to 27 cycles.
5. Artifact production by PCR is minimal within the range of 0.2 to 0.3μM of each primer concentration. With the above conditions, amplified fragment length polymorphism (AMR-FLP) obtained by polymerase chain reaction from the genomic DNAs of 4
Korean
families (one family with 8 offsprings and both parents, 3 families composed of each one child and both parents) were analyzed to test the applicability of this technique to paternity testing. The results obtained present reliable parent-child
relationships based on the correct genotypes of D1S80, without any undesired PCR products of abnormal length that are postulated as heteroduplex resulting from cross annealing or hybridization between the amplified target DNA.
|