The genome is the set of genes present in a cell. About 99.9% of the human genome is almost similar to each individual. But 0.1% of the variance in sequence defines an individual’s identity, making them different from each other.
The genome consists of both coding and non-coding region. The non-coding region is the repeated sequences that are inherited and do not code for the protein but make up most of the genetic DNA (deoxyribonucleic acid), whereas the coding region codes for proteins with a unique function.
The non-coding regions are also known as junk DNA or satellite DNA. Those sequences are used to correlate and differentiate individuals using techniques such as DNA fingerprinting, DNA profiling, and DNA typing.
Introduction and History
DNA fingerprinting is a molecular technique for determining the paternity and the suspects from crime scenes. This technique was discovered by Sir Alec Jeffrey in 1984 when he found some short tandem repeats (STR) or satellite genomes in seal meat with some similarities to humans.
Later, these STRs were used as genetic markers and produced a probe with short sequences aligned with those repetitive sequences. After the experiment, it was also discovered that these STR sequences vary in length for each individual. Although the bases of the sequences from a non-coding section of DNA in most living beings may be the same, its STR length differs from one individual to another.
DNA fingerprinting uses segments of the genome made up of tandem repeats of non-coding nucleotide sequences known as satellite DNA. Tandem repetitions are sets of copies of the same nucleotide sequence and may repeat once or more. For instance, the tandem repeat CG CG CG CG CG repeats the sequence CG several times.
The satellites are of three types depending upon the length, composition, and the number of tandem repeats, i.e., mini-satellite, micro-satellite, and macro-satellite.
Microsatellite, also known as short tandem repeat (STR), consists of 1 to 9 base pairs; for example, “3′-GATAGATAGATAGATA-5′ is the main STR repeated sequences makers and is repeated five to a hundred times at each microsatellite locus. Microsatellite does not have the exact location and is scattered randomly in the chromosome.
Mini-satellites, also known as variable numbers of tandem repeat (VNTR), may consist of 10 to 100 base pairs unit of length of repeated sequences and are found near the telomere. These units can repeat about two to a hundred times at the site of each mini-satellite.
Macro-satellite may consist of repeated units of several kilo-bases in length. These are the largest tandem repeats. D4Z4 and DXZ4 are some examples of macro-satellite.
During recombination, mutation of repeated sequences from both parents (1:1 ratio) causes the alternation of VNTR regions, making the child different from their parents. This principle is the root of DNA fingerprinting and genetic mapping.
Steps of DNA Fingerprinting
The process of DNA fingerprinting begins with the collection of samples, followed by DNA extraction which is then amplified and observed by electrophoresis or under X-ray.
Collection of specimen: The DNA sample is collected from body fluids such as blood, saliva, sweat, tears, tissues, and hair.
Extraction of DNA: DNA is extracted either by manual method or commercially available extraction kits. Two different methods further analyze the extracted DNA; polymerase chain reaction and restriction fragment length polymorphism.
Polymerase Chain Reaction (PCR) is a reaction that doubles the DNA copies in each cycle.
- After DNA isolation, the extracted DNA is subjected to PCR to obtain the number of copies of targeted sequences.
- Mathematically, the number of copies obtained can be calculated as, The number of copies = 2n, where “n” refers to the number of cycles. Over 20 cycles, there are precisely 1,048,555 copies of DNA.
- The main objective of performing PCR is to amplify the small amount of targeted DNA sequence. Agarose Gel Electrophoresis can help visualize the amplified DNA.
Restriction Fragment Length Polymorphism (RFLP) is a technique where a large section of DNA is digested using a restriction enzyme. This method is possible only on large DNA as short DNA may not contain the restriction site.
The restricted DNA is further subjected to electrophoresis, in which the band’s separation takes place based on the size of the sequences. The fragmented DNA is again transferred to the nitrocellulose membrane containing a radioactively labeled probe, and this probe aligns with the specific complementary VNTR region of separated DNA.
RFLP hybridization technique is commonly known as Southern Blotting. Next, the nylon membrane is washed to remove the excess probe. The nylon membrane is then exposed to X-ray to ensure targeted DNA’s bound with a probe.
The positive sample shows dark bands when exposed to X-ray, whereas the negative sample shows no bands with respect to the ladder. This process is also known as autoradiography. Lastly, the dark bands in the membrane (DNA fingerprint) are compared with the known samples.
Applications of DNA Fingerprinting
DNA fingerprinting is a widely used molecular technique with wide applications in different fields. Forensic analysis and determination of ancestral origin are the main areas of its applications. Following are some of the applications of DNA fingerprinting:
- To study the evolutionary relationship by analyzing the similar sequence between the individual.
- To examine the paternity as well as pedigree analysis.
- The criminal involved in the crime is easily investigated after matching the DNA from the crime scene with the suspects.
- DNA fingerprinting is also used in donor identification during an organ transplant.
- It is important in genetic counseling on genetic diseases and disorders.
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- Saad R (2005) Discovery, Development, and Current Applications of Dna Identity Testing. Baylor University Medical Center Proceedings 18(2): 130– 133.