DNA Typing

DNA or Deoxyribonucleic acid is the genetic/hereditary material that we carry inside our cells. Most of the organisms except some plant RNA viruses and retroviruses, all possess DNA as their genetic material. DNA could be nuclear DNA or mitochondrial DNA. DNA is basically two strands intertwined around each other that are made up of alternating sugar or deoxyribose,  phosphate group and nitrogenous bases.

The nitrogenous bases are classified as purines and pyrimidines, these are Adenine, Thymine, Cytosine, and Guanine. These nitrogenous bases are connected with each other through hydrogen bonds and are paired as Adenine – Thymine and Cytosine — Guanine. All of these three components and chemical bonds together form a double helix structure and encodes the biological information of an individual.

This biological information in the forms of codes is the most unique feature of an individual. But do you know it is just the 0.1% of the coding that makes all of us unique and the rest 99.9% of the DNA that we share is identical ,forming a common thread across the world? Humans have 3.2 billion base pairs that are distributed in 23 chromosomes.

And out of this 3.2 billion base pairs only 0.1% has an utility factor w.r.t DNA Typing or DNA fingerprinting. DNA typing is an umbrella term for studying genetic variations through different methods and its application for individualisation. 

Both the Nuclear DNA and Mitochondrial DNA are used to study genetic variation. Nuclear DNA tells us about the heredity of an individual whereas mitochondrial DNA specifically tells us about the maternal lineage of an individual as mitochondrial DNA is inherited from the mother only.

DNA Typing

DNA is also known as the biological blueprint of life while DNA typing is also known by various names such as DNA Profiling, genotyping or identity testing. DNA fingerprinting was developed by Dr.Alec Jefferys in the year 1984, he was the first geneticist  who noticed that in between the coding genes lies some remarkably variable sequences of DNA which do not contribute to the functions of genes and  are repeated within genes. These non-coding, highly variable sequences are known as introns or minisatellites,discovered in 1980. Further, he comprehended that each individual (except identical twins) has a unique pattern of minisatellites.

Ergo, DNA typing is nothing but the method of isolation, amplification and identification of these variable non coding tandem repeats within the base pair sequence of a DNA and finally creating a DNA fingerprint of any individual. This identification could be further used in individualisation, identification, paternal testing, maternal testing, parentage testing, linking crime and criminals, identification of victim, identification of skeletal remains found in archeological sites, connecting skeletal remains with a particular species in anthropology etc.

Sources of DNA

The sources of DNA are biological in nature. These are:

• BLOOD
• SEMEN
• SALIVA
• TEETH
• HAIR 
• URINE
• TISSUE
• BONE

Techniques used for DNA typing

• RFLP – Restriction fragment length polymorphism is a technique that exploits the variation in homologous DNA sequences known as polymorphism. The phenomenon of polymorphism is exploited in RFLP analysis in which a DNA sample is digested into fragments using restriction enzymes and then separated using gel electrophoresis.

• STR – STR or short tandem repeats are small sections of DNA  sequences that consist of 3-5 base pair repeats. A number of STRs are located and compared simultaneously. USA uses 13 STRs for identity in CODIS database, UK uses 11 STRs in its NDNAD. STR can be used in PCR techniques.

• Y- Chromosome Analysis- Y- STRs are inherited paternally and thus can link an individual to his paternal male relatives. Y chromosome analysis is also used to identify the sex of the biological samples. It is based upon the fact that male alone have Y chromosomes in addition to X chromosomes.

• Mitochondrial DNA Analysis– Mitochondrial DNA links mother and other maternal relatives. It varies from nuclear DNA , human mitochondrial DNA contains 2-10 units of double stranded circular DNA. Mt DNA profiling is coming up as an important tool to establish the identity or non identity of an individual from hair shafts, teeth and bones even when they are old.

Procedure of DNA Typing

The DNA typing procedure mainly constitute of the four following steps:

1. EXTRACTION-  We all are aware about the location of DNA i. e inside the nucleus of our each and every cell. To extract the DNA for further processing we need to break open the nucleus and release the DNA. The most common extraction procedure is Organic extraction using Phenol, Chloroform and Isoamyl Alcohol. It is manual in nature and takes around 3 hours.. Apart from these we also use Chelex extraction procedure, FTA paper extraction , running samples on robotic systems such as Maxwell16 , Solid Phase method etc. The extracted DNA from samples like blood is often stored with preservatives like EDTA.

2. QUANTITATION –  This process of quantitation is done for assessing the quantity and quality of the DNA extracted. Determining the amount of DNA extracted from the sample is essential for success in the amplification stage. The amplification procedure requires a specific amount of sample DNA. This process of quantitation could be done using Quantifiler DNA Human Quantification kit and further running the samples on an instrument known as Sequence detection system ( ABI PRISM 7500).

3.  AMPLIFICATION –  The process of Amplification is one of the most important steps in DNA typing specially for forensic purposes as the DNA samples found at crime scenes are often very limited and contaminated in nature. The amplification is accomplished through Polymerase Chain Reaction technique.The PCR technique was developed by Karry Mullis. There is a selective amplification of  a target DNA sequence within  a collection of DNA sequences. The PCR cycle includes Denaturation, Annealing and Extension. These processes involve precise heating and cooling of the samples in a thermal cycling pattern. After these 28 cycles millions of copies of a specific sequence of DNA can be made within hours. There are many variants of PCR such as Multiplex PCR, Asymmetric PCR, Allele Specific PCR, Cold PCR, Touch down PCR etc.

4. ELECTROPHORESIS –  The electrophoresis process is the last step of DNA typing. Capillary Electrophoresis is the type of electrophoresis that is done for separation of DNA molecules. The amplified DNA sequence obtained after the PCR process is a mixture of amplified DNA molecules that needs to be separated and distinguished from one another. DNA is a negatively charged molecule and if an electric current is applied it migrates towards the positive anode of the thin capillary which is  filled with a gel-like polymer. The DNA strands are separated  on the basis of size as the smaller DNA molecule will migrate through the polymer faster than the larger molecule. 

The sequencing of the DNA is done with the help of GENETIC ANALYZER MACHINE. Finally, the DNA profile of the separated DNA is produced in the form of an electrogram or a graph with 2 peaks for each locus. The graph obtained for questioned and reference sample are then compared with each other, if both the graph shows equal peaks and no. of repetitive sequence at particular loci then it shows common origin, if not then the origin is uncommon and if there is any drop or only one peak matches and the other is not obtained then in such cases the result is said to be inconclusive.

Applications of DNA Typing

DNA typing has many applications in today’s world and since its inception  it has proved to be a groundbreaking technique in the field of forensics, archeology and anthropology. DNA is one such evidence or identity of an organism that does not degrade easily even in the highest temperatures. DNA has been isolated from organisms whose presence dates back to thousands of years ago whether of animal or human origin.

This property of DNA is now also being used to isolate drugs from Dust present in a room since we shed our cells regularly, it gets accumulated on the floor in the form of dust. This is one of the most novel research being done by the forensic fraternity.

Various DNA banks are also being made in different countries of their countrymen whether involved in a crime or not. Apart from this DNA banks related to animals are also being created by various wildlife protection agencies to aid in investigations related to wildlife crime such as poaching and trafficking of endangered and exotic wild animals.

Amongst the wide variety of applications. These are some of the most useful and noticeable applications of DNA typing:

• Solving crime: Matching of suspect with evidence, Matching of biological evidences from suspect and victims in a sexual assault case.
• Identification of bodies: In calamities like aeroplane crash, avalanches, arson, bomb blasts or mass murder dead bodies are identified using DNA typing.
• Paternity testing:  Paternity testing is done for identification of the biological father of an individual in paternity disputes.
• Missing person investigations: Missing person investigation is done by analysing the skeletal remains discovered and connecting it with their relatives DNA.
• To maintain a database of convicted criminals.
• To solve immigration and inheritance disputes.
• Ancestry and racial identification.
• Diagnosis of genetic disease.
• Identifying remains from wars.
• Identifying remains from archeological mounds and sites.

FAQs about DNA Typing

References

1. https://dps.mn.gov/divisions/bca/bca-divisions/forensic-science/Pages/dna-procedures.aspx#:~:text=The%20DNA%20testing%20process%20is,%2C%20amplification%2C%20and%20capillary%20electrophoresis.
2. Jordan D and Mills D (2021) Past, Present, and Future of DNA Typing for Analysing Human and Non-Human Forensic Samples. Front. Ecol. Evol. 9:646130. doi: 10.3389/fevo.2021.646130
3. Butler, J. M. (2009). Fundamentals of forensic DNA typing. Academic press.