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.
Identification of any person in an important process for the crime scene investigation and other property claims.
The finger or thumb fingerprinting method was the gold standard method for personal identifications since 1984. The fingerprint of an individual is unique from another person. However, it can be changed by surgeries which creates a major haphazard problem in the identification process.
However, the era of personal identification was changed after the discovery of the DNA fingerprinting method.
On the other side, the DNA fingerprint of an individual is unique, although 99.99% DNA is the same in all human, 0.1% difference is sufficient to make someone so unique.
No two person in this world has the same genetic composition.
One of the most interesting things about the DNA fingerprinting is that the DNA remains the same in all the body parts, tissue and cells in our body (except germ cell). Even, after the death of the individual, the DNA remains as well.
It can remain stable even after 1000 years.
The human DNA is full of repeated sequences, the numbers of the repeat and nature of repeats are different among different persons.
Based on this data the DNA fingerprinting technology is developed and it is a big blow in the forensic industry.
Though the PCR based method for DNA fingerprinting is accurate, after the discovery of real-time PCR and DNA sequencing, the method becomes even more powerful.
The molecular tools available nowadays creates 100% accurate pattern of DNA fingerprinting of an individual.
The DNA fingerprinting method is based on the characterization of the region of DNA which is highly polymorphic. In the present article, scientifically we are going to explore each and every detail of DNA fingerprinting.
The content of the article is,
- History of DNA fingerprinting
- The role of non-coding regions in DNA fingerprinting
- Sample type for DNA fingerprinting
- The process of DNA fingerprinting
- DNA fingerprinting techniques
- RFLP based STR analysis
- PCR based analysis
- Real-Time PCR analysis
- Application of 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 the structure and function of DNA read the article: The structure and function of DNA
DNA fingerprinting technique is originally developed by a British scientist Alec Jeffreys in 1984. Dr Lalji Singh is called a father of DNA fingerprinting in India.
Jeffrey used the technique of RFLP for the analysis of a DNA profile. In fact, not only RFLP, it is a combination of RFLP and autoradiography.
Before going into the actual process of DNA fingerprinting we have to understand how the satellites of chromosomes involved in the DNA fingerprinting.
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. However, 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, this 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.
Some of the related articles,
The role of a non-coding region in DNA fingerprinting:
Any protein is encoded by the gene and that is why gene exists in nature. It codes for protein. It replicates, transcribed into mRNA and finally translated into the protein.
The non-coding region of the genome cannot code for any particular protein. It is just a bulk of repetitive sequences hence it is known as junk DNA.
However, the junk DNA involves in one of the important phenomena in nature, the gene expression.
The DNA sequences present into the junk DNA regulates the expression of a gene in particular tissue or in a particular organ.
95% of human DNA is junk repetitive DNA sequences. The longer repetitive sequences are called as VNTR and shorter repetitive sequences are called as STRs.
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).
By digesting the one particular VNTR region with one particular restriction endonuclease in three different people, the results on the gel look like this,
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
DNA can be isolated from any of the biological samples.
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.
The process of DNA fingerprinting:
The DNA fingerprinting process starts with DNA extraction.
For all the genetic applications, DNA extraction is one of the most crucial steps. We have to extract a good quality DNA with high yield.
However, we strongly recommend using ready to use DNA extraction kit for DNA fingerprinting.
If you want to extract DNA from the plant tissue, use the CTAB plant DNA extraction method.
The purity of DNA must be ~1.80 and the quantity around 100ng.
If DNA is not purified or not extracted well purify the DNA using the DNA purification kits.
After that, quantify the DNA using the UV-Visible spectrophotometer. And perform one of the following method listed below,
DNA fingerprinting techniques:
The major types of 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 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 are 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.
The radio-labelled probes are applied for the hybridization. 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.
The RFLP method is more accurate than the DNA if a large amount of sample is given.
Nonetheless, this technique had several limitations.
The method is tedious, takes a lot of time and hence it is costly. Also, a large amount of DNA required which makes it restricted for forensic analysis.
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.
It is faster and more accurate.
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 primers are designed based on the sequence data.
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).
The amplified DNA fragments are analyzed on the agarose gel and based on the size of the fragment, it migrates in the gel and gives a different banding pattern.
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.
The PCR method can identify different fragments of STR or VNTR but still, we can not count the exact number of the repeats of a suspect.
3. Real-time PCR analysis:
Real-time PCR gives 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.
We can calculate the number of repeats present into different samples.
Graphical representation of different DNA sample. After the amplification, DNA sequencing gives more accuracy which determines the sequence amplified and the number of the repeats present into the sample.
Nowadays the DNA fingerprinting method is more reliable on the DNA sequencing because it gives accurate results.
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 standard 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 the 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.