The processes reduced the time frame which was 6 to 8 weeks to a few hours now. There are various DNA markers and they have been divided into four different quadrants depending upon the power of discrimination and speed at which they can be analyzed.
ABO Blood group system
One of the earliest methods of analysis was based on ABO Blood group system. Most (80%) of the population will be either type O or type A. Exclusion of an individual from the crime is possible but doesn’t help in finding the culprit. Blood will get agglutinated or clump when blood from different people were mixed together.
Multilocus RFLP Probes
Multilocus probes provided a lot of information and are highly variable, but the process is very expensive and involved intensive labor.
STR (Short Tandem Repeats)
Short tandem repeats (STR) can be used to analyze three or more loci during the same time. They produce highly discriminating results even from degraded samples.
Mitochondrial DNA (mtDNA)
Mitochondrial DNA (mt DNA) has low power of discrimination, used in analyzing degraded DNA samples and also in samples where nuclear DNA is not present (hair shaft, putrefied bones and teeth).
Forensic Protein profiling
Forensic Protein profiling was one of the methods that were followed before DNA testing. Aminoacids sequences in some of the proteins vary in the human population and were used for protein profiling.
Restriction Fragment Length Polymorphism (RFLP)
RFLP uses restriction enzymes to cut the DNA and the fragments of DNA are transferred to the nylon membrane. The hybridization of the membrane to the labeled DNA probe determines the size of the fragment. The RFLP process took several weeks to complete.
The restriction enzymes used were HaeIII, HinfI, and PstI. The most common one was the HaeIII. It requires more amount of DNA and it is difficult to analyze the DNA using degraded samples. Long-Range PCR amplified the VNTR region using Taq polymerase and a thermostable proofreading enzyme. The first available multiplex STR Kit was from Promega Corporation and consisted of the STR loci CSF1PO, TPOX, and TH01.
They are referred to as CTT Triplex. Silver staining STR kits were first commercially available from Promega Corporation. It was less expensive and required only silver nitrate and gel electrophoresis box.
Fluorescently tagged STR kits
Fluorescent tagged STR kits are used now to identify the length polymorphism of the PCR products using Fragment analysis in genetic analyzers. A small amount of DNA is required for this process and even profiling can be done in degraded samples.
PCR products obtained from the conventional PCR method can be sequenced using Chain termination or Sanger sequencing method by the selective incorporation of dideoxynucleotide triphosphate (ddNTPs) to the sequencing reaction in addition to deoxynucleotide triphosphate.
Dideoxy nucleotides lack 3′ OH group, unlike deoxynucleotides which aid in the formation of phosphodiester bonds between nucleotides (for details please check the blog of Sanger Sequencing).