Question

how is the sequence specific primer (SSP) PCR and multiplexing method used to analyze HLA type carried out, what are the steps?

Answer 1

SSP (sequence specific primers) are short primers which are specific for particular locus in the genome. Hence it cannot amplify any other sequence in the genome. HLA, the human leucocyte antigen is a highly polymorphic region in the genome. Many different alleles are present in different individual for one particular gene. 2 class of HLA genes are present. It indicates that one single primer is amplify a single allele or a group of similar kind of alleles. Because of sequence specificity of primers, large numbers of primer pair are required to perform the experiment. So it is not possible to perform single experiments for each set of primers.

Multiplex PCR provides flexibility for performing multiple experiments in a single tube. The steps are as follow.

DNA of patient as well as donor is extracted. The extracted DNA is quantify and the quality is measures.

Now the PCR reaction is set up for group of 4 or 5 pairs of primer as one reaction.

PCR is run using stander PCR protocol.

The result is analysed on agarose gel electrophoresis.

Here the primers are tissue specific, hence a single primer can amplify a single allele. The bands are observed as per the amplified locus. If up to 90% of locus will be matched between patient and the donor, than it the organ implantation or bon marrow transplantation is possible.