Length Heterogeneity Polymerase Chain Reaction
Length Heterogeneity Polymerase Chain Reaction (LH-PCR) reveals the length heterogeneity of amplicons. The procedure indicates the natural variations of insertion and deletion of nucleotides. LH PCR exposes the allelic profile pattern of DNA in a particular loci. Species-specific differentiation is possible using this method. The procedure is rapid simple and reproducible.
LH-PCR/Fragment Analysis is an amplification-based technique using forward labeled primer and reverse primer. The PCR is similar to conventional PCR except the forward primer is labeled with a suitable fluorescent dye. Agarose Gel Electrophoresis is an essential step after amplification and DNA bands can be viewed under transilluminator. After amplification, amplicons along with Hi-di formamide (denaturing agent to keep the DNA single-stranded) and size standard should be loaded onto the Genetic analyzer in order to size the amplicons. The raw data obtained are read using Fragment analysis software in the computer attached to the Genetic analyzer.
Allelic profile pattern is represented in the above diagram. Fragment size (base pairs) can be read from the horizontal axis and Relative fluorescent units can be read from Y-axis.The peak intensities (in relative fluorescent unit- rfu) are directly proportional to the concentration of the DNA template.
Wild type is an allele without any insertion or deletion of nucleotides. In LH-PCR, the insertion/deletion of nucleotides can be identified within the same species in a specific loci. Microbial changes in soil can be identified by testing the soil obtained from both graveyard soil and control soil. This procedure can lead investigators to the identification of criminal or missing persons by further analysis using specific assays for the detection/findings of buried bodies in the soil.