Watson and Crick postulated that DNA consists of two strands that twist around each other to create a right-handed helix. Anti-parallel strands That is, one strand's 3' end is connected to another strand's 5' end. The backbone of the structure is composed of pentose sugar and phosphate groups, with nitrogen bases layered inside. Complementary base pairing is another intriguing property of DNA. Purine and pyrimidine nitrogen bases are paired here. Purines are adenine and guanine, while pyrimidines are cytosine and thymine. Hydrogen bonds help to hold those base pairs together. The genetic information required to construct and control the cell is stored in DNA. When cells multiply, DNA is duplicated. So DNA does not produce a structural role as other macromolecules.
What is DNA Fingerprinting?
VNTR, a genetic marker, is used in this technique. Variable Number Tandem Repeats (VNTR) is a term that refers to the number of times a pair of the genome contains a lot of non-coding DNA. The number of repetitions varies widely. Even homologs of the same chromosome can have different characteristics. As a result, VNTRs are used as markers for individual identification, a process known as DNA fingerprinting or DNA profiling. Markers allow effective selection of recessive alleles of desired traits in heterozygous status, as morphological features are insufficient to identify individuals. A DNA fingerprinting test was used for the first time in India in June 1989 to settle a long-running paternity case in Madras. For theft detection and paternal disputes, fingerprint evidence is used. Blood, saliva, hair, nails, sperm, sweat, and cells are common sources of DNA extraction.
Figure 1 - Steps in DNA Fingerprinting
DNA Fingerprinting Methods
RFLP-based fingerprinting and PCR-based fingerprinting are the two methods used. The DNA primers used in the genomic fingerprinting method correspond to naturally existing repeating sequences in bacteria.
RFLP Based fingerprinting
Restriction enzymes are employed here. Restriction sites cut double-stranded or single-stranded DNA. Different restriction enzymes recognize and cut DNA sequences at different locations. EcoRI, EcoRII, BamHI, TagI, HindIII, and others are restriction enzymes (RE). Bacterial enzymes are thought to have evolved as a defence mechanism against virus invasion. Sticky ends and blunt ends result from restricted digestion. RFLP is a technique that uses patterns derived from DNA cleavage to differentiate organisms. When DNA is digested, the length of the fragments produced will differ if the distance between sites of cleavage of a restriction endonuclease differs between two organisms. The similarity of the generated patterns can be used to distinguish between species and strains. When considering the procedure, we must first extract DNA before cutting it with RE. There are many DNA fragments as a result. Electrophoresis on an agarose gel can help separate these pieces. All bands appear as a blur and are difficult to discern; individuals cannot be compared. As a result, a subset of bands with which to compare is required. To identify a sample, use the Sothern Blotting approach.
Single strands of DNA are blotted to a nitrocellulose membrane in the Sothern Blotting technique. Probing is the last step. Hybridizing with a complementary sequence with a radioactive label is what probing is all about. After that, expose all bands with the probe to an X-ray film. This strategy has both benefits and drawbacks. Stable and repeatable results, ease of use, and a shorter time frame are all advantages. When used as a dominant marker, the disadvantages include the fact that identical length fragments with varied nucleotide makeup may appear on the gel as homologous, resulting in incorrect results.
PCR Based Fingerprinting
Kary Mullis and his colleagues developed it in 1985 at the Caus Corporation's Henry Elrichs Laboratory. Conventional PCR, Real-Time PCR/quantitative PCR, and RT-PCR are the three forms of polymerase chain reaction (PCR). As markers, there are short tandem repeats (STR). The length of STRs might range from 10 to 100 bp. If homologous chromosomes are similar, they are homozygous; if they are different, they are heterozygous. As a result, allelic/genotype data can be shared. The flanking sections are preserved, even if the repeat sequences are highly varied. This means that the bordering regions do not differ from person to person. The flanking regions are used to create locus-specific primers for PCR amplification. We can readily detect repeat variation in terms of fragment length following PCR if we use these primers.
Each forward primer is coated with a very sensitive fluorescent dye that may be detected by a LASER sensor. A sophisticated genetic analyzer and DNA sequencer are required. Individual identification requires the discovery of human genetic variation. DNA fingerprinting has progressed to the point that it can positively identify a person. Under the provided temperature profile, DNA amplification occurs at its optimum. With the use of UV light and DNA staining, the final product could be seen. In traditional PCR, the intensity of amplified samples is evaluated after the amplification reaction is done, which is usually after 30–40 cycles. The amount of template contained in the sample is back-calculated using the final intensity. The observed fluorescence in quantitative PCR is proportional to the overall amplicon quantity.
The technique's advantages include great reproducibility, the ability to multiplex distinct microsatellites in PCR, a wide range of applications, automation, and the use of small amounts of template DNA (10–100 ng each reaction). The high mutation rate in microsatellite loci makes it unsuitable for higher-level systematic investigations, and stutter bands may make precise polymorphism scoring more difficult.
Applications of DNA Fingerprinting
DNA fingerprints can be utilized in forensic instances such as homicides, paternity disputes, maternity testing, mutilated remains identification, pedigree lineage tracking, diagnosis of inherited diseases etc. Every band in a child should come from either the mother or the father when it comes to family disagreements. It is far easier to demonstrate the bad side than the good side in this situation. Bands must be linked with the accused in criminal/forensic proceedings, as well. DNA databases are particularly valuable in forensic investigations. DNA databases are maintained in the majority of developed countries, such as the United States, the United Kingdom, and China. DNA-based molecular markers are now used in horticulture, in addition to traditional breeding efforts.
Gel electrophoresis is the approach that we utilized, which is now widely used in the forensic sector. DNA may be tested from any sort of evidence (hair, blood, skin, etc.) and is reasonably priced. However, because the gel might be tampered with and produce false findings, we use Sanger Sequencing and Polymerase Chain Reaction to corroborate the results (PCR). (2015) (Aziz et al.) DNA technology is now widely used throughout the world, and it determines the sentence for all crimes without regard for the circumstances. DNA is a divine sign that is unique to every person.
SIFS India is the best-renowned laboratory to provide its services for analysis of each type of evidence ranging from Fingerprint to Document, Blood group testing to DNA Fingerprinting and Insurance Investigation to accident reconstruction. They also provide their services for fingerprint verification for PCC from different countries around the globe. At the same time, SIFS India is also a recognized and beloved institution by students and forensic fraternity from India and other countries for their best and renowned expert forensic training by the best faculty. Along with training and teaching, they also provide educational assistance to the students and lifetime learners of Forensic Science in form of Forensic Quiz Series, Expert Talks, Workshops, International Conferences as well as other Forensic Events from time to time on an online platform with diverse personalities of forensic science. So for any assistance from evidence analysis to training, learning, and certification, SIFS India is the one-stop solution for all forensic needs.
Learning at SIFS INDIA
To learn more, visit the Sherlock Institute of Forensic Science India where the team of expert professionals and certified faculty provides you with a great opportunity to learn forensics by attending different forensic events organized from time to time. Also, you can learn by registering yourself in forensic courses and training programs offered by SIFS India both in online and offline mode. You can also visit our YouTube channel for all the events recoding.