DNA fingerprinting technique and its applications
“Identification of individual on the basis of its DNA data or DNA profile is called as DNA fingerprinting.” DNA fingerprinting has many other names,
DNA profiling, DNA testing, DNA analysis, Genetic profile, DNA identification, genetic fingerprinting and genetic analysis are some of the common names used for DNA fingerprinting,
History of DNA fingerprinting:
Since the discovery of DNA in 1953 by Watson and Crick, genetic science becomes more advanced and accurate.
For more detail on DNA read the article: The story of DNA
DNA fingerprinting technique is originally developed by a British scientist Alec Jeffreys in 1984. Dr. Lalji singh is called as a father of DNA fingerprinting in India.
He used the technique of RFLP for the analysis of DNA profile. In fact, not only RFLP, it is a combination of RFLP and autoradiography.
In Genetics, the satellites are repetitive DNA regions which can protect the whole chromosome and locate in centromere and telomere regions. Mainly two types of satellite regions are present in human genome based on their repeat sequence nature: minisatellite and microsatellites.
Minisatellite region contains repeated DNA sequences of 10 to 60 bp long and repeated 5 to 50 times in a genome. VNTRs are the best example of minisatellites. The repeat numbers vary from individual to individual.
It is a highly variable region and is unique for each individual. minisatellites are GC rich region. it is found mostly in the telomeric region (90% sequences).
Microsatellites are smaller than minisatellites. It is ranging from 1-6 bp long and repeated 5 to 10 times. Generally, these microsatellites are STRs and SSRs.
It is highly repeated sequences, varies from individual to individual and located on the telomeric region.
We have covered the entire article on VNTR and STR in genetic marker, for more detail understanding of genetic marker read the article: Genetic markers
Interestingly, the first microsatellite was discovered by Jeffreys and his coworkers in 1984. The name microsatellite was given by Litt and Luty in 1989.
An interesting story
Everyone thinks that the name satellite is given because the sequences are located on the telomeric region of the chromosome. However, the conception is not true.
The satellite DNA name is given to this sequence because of their nature of separation in the centrifugation process.
Quantitatively the large portion of the human genome is made up of repeated sequences and hence it appears as a thick prominent layer on the top of the test tube after the centrifugation. So the name is given as a satellite DNA.
Now get back to our original topic,
Jeffreys and coworker identified the first microsatellite. The DNA fingerprinting was performed through RFLP & autoradiography.
Jeffrey extracted DNA from the sample and digested it with the help of the restriction endonuclease. The fragments are separated on the basis of size by agarose electrophoresis.
Then the DNA is transferred to the nylon sheet and proceed for the southern blot with labelled probes.
The results were analyzed with the help of the X-ray film. This was the first method adopted by the scientist for the identification of short tandem repeats from human samples.
Before going further on different techniques of DNA fingerprinting, lets first understand the correlation between tandem repeats and DNA fingerprinting.
Tandem repeats and DNA fingerprinting
“Tandem repeats are the sequences which are located one after another into the genome.”
It varies from individual to individual, for example, if a person A has 45 VNTRs (with 20bp) and 9 STRs (with 5bp), The possibility of having this same number of repeats for a specific VNTR and STR in another individual is almost negligible. Nonetheless, in the case of monozygotic twins, it is possible.
The monozygotic twins are developed from the single type of embryo (due to the splitting of an embryo), so the chance of having the same type of VNTR and STR profile is higher.
For normal individuals, the chance of having the same DNA profile is 1 in 10,000,000,000,000( the total world population is 7,600,000,000, imagine the possibility).
So the DNA fingerprinting technique is unique and highly reliable for the identification of individual in case of paternity verification or criminal identification.
Types of a sample for DNA fingerprinting
Generally, DNA can be extracted from any of the biological samples. Buccal smear, saliva, blood, amniotic fluid, chorionic villi, skin, hair, body fluid and other tissue are the major types of the sample used for DNA fingerprinting.
For the suspected criminal verification buccal smear is routinely used. Buccal smear is easily available and it is non-invasive. The blood is the best source of a sample if the buccal smear is not available.
DNA fingerprinting techniques:
The major types of DNA fingerprinting techniques used for the DNA fingerprinting are:
- RFLP based STR analysis
- PCR based analysis
- Real-Time PCR analysis
1. RFLP based STR analysis:
- Restriction fragment length polymorphism was the first method used in the analysis of DNA fingerprinting.
- DNA is extracted with the help of enzymatic extraction method from the sample.
- The DNA is now digested with the help of restriction endonuclease which generates different DNA fragments based on its length.
- The digested DNA is separated with the help of agarose gel electrophoresis. Under the influence of the current, the fragments are migrated and different fragments of DNA is observed under the UV transilluminator.
- Now the fragments are transferred to the nitrocellulose paper and proceed for southern blot analysis.
- The DNA is chemically denatured and permanently fixed on the nitrocellulose paper.
- Now the radio-labelled probe is applied for the hybridisation. The radiolabeled probes are complementary to repeat sequences hence it will bind to it.
- The probes and denatured DNA on nitrocellulose paper are now incubated for the hybridization. Every time when a probe finds its complementary sequence it will bind to it.
- The non-bounded probes are removed from the site of hybridization by washing because it will hinder in autoradiography.
- Now our hybridized blot is ready and it is exposed to the X-ray film.
- The radiolabelled probed which binds to its complementary sequence have appeared as a fluorescent band under autoradiography.
Nonetheless, this techniques had several limitations. The accuracy is fewer. It required a large amount of DNA for additional restriction digestion process, therefore, it is not suitable for crime scene investigations. At a crime scene, the amount of sample is very less.
2. PCR based analysis:
- PCR based gel electrophoresis analysis is one of the most popular methods of DNA fingerprinting.
- It is simpler than RFLP-autoradiography and gives accurate results than the traditional techniques.
- Now we have numbers of known STRs and VNTRs are present in the NCBI database. We can choose any of the STR or VNTR of our interest.
- After the choice of specific repeat sequences, the primer is designed based on the sequence.
- Now DNA is extracted from the given sample and the PCR gives us millions of copies for that particular DNA sequence.
- Simple PCR based agarose gel electrophoresis is enough to examine the result (if you are an expert).
- However, PCR based gel electrophoresis is mostly used for VNTR analysis because of their length.
- STRs are smaller in length so it is difficult to distinguish two fragments into a gel. In contrast, VNTRs are larger fragments and gives beautiful distinct band pattern in agarose gel electrophoresis.
Capillary gel electrophoresis gives the best result for both STR and VNTR because it can discriminate even the most nearest DNA fragments.
3. Real-time PCR analysis:
Real-time PCR gives a real-time data of amplified fragments, the length of the amplified fragment and the accuracy of amplification in the form of a real-time graph.
The process is quick, reliable and accurate therefore it is widely adopted in criminal identification. Even if a small quantity of DNA is enough for the analysis.
- In RT PCR fluoro labelled probes are used, Instead of simple complimentary primers. however, the probes are not as long as it is used in the southern blot.
- Once the fluoro labelled probe is bound to its complementary sequence, it will give a signal to the machine which monitors the amplification process of that particular fragments.
- Based on the fluorescent signals it creates different peaks of a graph for different numbers of the repeat.
- No need to run electrophoresis so this technique is so fast and very reliable.
Application of DNA fingerprinting:
Identification of individuals and Paternity verification
In case of confirmation of one’s identity, there is no other better option than DNA fingerprinting. The sample is taken from the person, DNA is extracted for the analysis and his or her DNA is matched with parents for identification.
Vice verse the parents are even verified using DNA fingerprinting.
Maternal cell contamination
One of the major drawbacks of prenatal diagnosis is maternal cell contamination. The amniotic fluid or CVS sample contains the maternal DNA or maternal tissue.
Hence the DNA become contaminated or the maternal DNA is extracted along with the fetus’ DNA which gives false result in the investigation.
A combination of VNTR and STR markers provides a great advantage in routine maternal cell contamination detection.
For more detail on Prenatal diagnosis read the article: The revolutionary non-invasive prenatal diagnostic technique in emerging medical science: cffDNA
Crime scene investigation
One of the important application of DNA fingerprinting is its role in crime scene investigation or criminal identification.
The sample is collected from the site of the crime. The sample could be saliva, blood, hair follicle or semen.
DNA is extracted from the sample. Another sample is collected from the suspect and STR marker-based analysis profile is used for the identification of the criminal.
Different countries have different criteria for the use of STR. In India we are using 11 STRs, in USA total 13 STR markers are used, in UK 11 markers are used for the forensic analysis.
As the number of STR markers are increased the accuracy of the result is increased.
Identification of blood relatives
No two individuals are genetically identical. Hence for identification of blood relatives, DNA fingerprinting is one of the most adaptive technique.
My experience of DNA fingerprinting
I was worked in a prenatal lab for identification of autosomal recessive disorders. Instead of following the stander protocol with STR or VNTR, I had prepared our own combination of markers.
We selected 4 VNTRs, 3 STR, 1 mitochondrial DNA marker and 1 Y chromosome specific STR for maternal cell contamination.
Mitochondrial DNA is additionally a great tool for identification of individuals. As different cells have different numbers of mitochondria, the number of mitochondrial DNA also varies from individuals to individual.
Y specific STR is as important as normal STRs because it is helpful in distinguishing males from female and it is a primary line of DNA fingerprinting analysis in crime scene investigation.
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Article written by: Tushar Chauhan
Article reviewed by: Binal Tailor