“A genetic technique or method of identifying individuals using bodily samples such as blood, saliva or hair is referred to 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 for DNA fingerprinting.

The present method is employed so often in criminal verification and crime scene investigation. However, it’s applicable in other biological processes too.

Not only humans but two different organisms can also identify using the DNA fingerprinting method.

Since, 1984, it’s a gold-standard method for personal verification. Because of the unique pattern of DNA two persons differ biologically. And this mechanism is the basis of the present method.

The DNA is a basis of life, encodes proteins and regulates gene expression. It is made up of sugar, phosphate and nitrogenous bases and located on chromosomes.

In the present article, we are going to talk about DNA fingerprinting and how it is done.

The process of it and different techniques are our major focus. we’ll discuss everything scientifically and in-depth. So let’s start the story,

What is DNA fingerprinting?

The era of personal identification changed after the discovery of the DNA fingerprinting method.

Notably, approximately 99% of our DNA is similar. 0.1% difference is sufficient to make someone so unique. We are using this 0.1% of DNA for DNA profiling known as DNA fingerprinting.

There is a reason, why only DNA is used,

DNA remains the same in all the body parts, tissue and cells (except germ cell). Even, after the death of the individual, it remains as well. It can live stable even after 1000 years.

As we know, 97% of our genome is non-coding, repetitive and junk. Only 3% of DNA are genes and encodes proteins.

Also, the junk region is highly polymorphic- different between organisms. Hence the Repetitive polymorphic property of the DNA is used in the present method.

The number and nature of repetitive DNA vary among individuals. This is the basis of DNA fingerprinting.

Broadly, the method is as stated,

The DNA is isolated from the bodily sample. Digested or amplified using restriction digestion or PCR. Using either agarose gel electrophoresis or sequencing, the DNA fragments are separated and identified.

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 fingerprint of individuals.

Definition:

“Using PCR, DNA sequencing or restriction digestion, the unique DNA pattern of organisms created, analysed and used for verification is called DNA fingerprinting.”

History:

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 was originally developed by a British scientist Alec Jeffreys in 1984. However, Dr Lalji Singh is known as a father of DNA fingerprinting in India.

Using the restriction digestion length polymorphism, Jeffrey created the first DNA profile. It was the combination, RFLP and autoradiography.

Now here we have to understand some basics before going further. First, let’s understand what are the satellite regions of chromosomes.

Satellites DNA:

In Genetics, the satellites are repetitive DNA regions, located on telomeres and centromeres. It protects the chromosomes from replication error, actually. 

Note one thing here, these satellite DNAs are non-coding.

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. 5 to 50 repeats of it are present in our genome. For example, VNTRs.

It is highly variable (polymorphic), unique and GC-rich sequences. As we said above, It is found mostly in the telomeric region (90% sequences).

Microsatellites are smaller than minisatellites. It’s ranging from 1-6 bp long and repeated 5 to 10 times. For example, STR and SSR. Read more on STR- short tandem repeats.

One more time, like the VNTR, it’s highly variable, non-coding and telomeric sequences.

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

The first microsatellite was discovered by Jeffrey and his coworkers in 1984. Although, 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. This conception is not true.

image credit: www.fhcrc.org

The satellite DNA name is given to these sequences 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. On the basis of size, DNA fragments were separated on an agarose gel. 

Then he transferred DNA 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 identification.

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,

  1. DNA sequencing
  2. What is Restriction Digestion and how to do it?
  3. Agarose gel electrophoresis

The role of a non-coding region:

Any protein is encoded by the gene and that is why gene exists in nature. Through replication, transcription and translation, DNA becomes doubled and forms protein via mRNA.

The junk DNA can’t form any proteins though, it helps in regulating gene expression. It’s one of the important phenomena in nature hence non-coding DNA is as important as genes.

Loosely packed DNA sequences present into the junk DNA regulates gene expression in cells, tissue or organ

VNTRs and STRs: 

“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.

Still, monozygotic twins ain’t similar biological. Read this article: Do Identical Twins Have The Same DNA?

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).

Difference between VNTR and STR: 

VNTR 

STR 

Variable number of tandem repeats  Short tandem repeats 
A type of minisatellite  A type of microsatellite 
Consists 10 to 60 bp Consists 1 to 6 bp 
10 to 1000 repeats in a genome  5 to 200 repeats in a genome 
Produce heterogeneous array  Produce homogeneous array 

By digesting the one particular VNTR region with one particular restriction endonuclease in three different people, the results on the gel look like this,

The agarose gel electrophoresis results of three different individuals.

This is the basic fundamentals behind DNA fingerprinting technology. Now, let’s understand the actual process.

Steps:

  1. Collection of a biological sample- blood, saliva, buccal swab, semen or solid tissue.
  2. DNA extraction
  3. Restriction digestion or PCR amplification
  4. Agarose gel electrophoresis, capillary electrophoresis or DNA sequencing
  5. Analysis of results

Process of DNA fingerprinting:

Sample collection, DNA extraction, digestion or amplification and analysis results are major steps.

Sample types: 

DNA can obtain from any bodily sample or fluid.

Buccal smear, saliva, blood, amniotic fluid, chorionic villi, skin, hair, body fluid and other tissues are the major types of the sample used for DNA fingerprinting.

In criminal cases, a buccal swab is taken routinely. It’s available easily and the sample collection method is non-invasive.

However, if not maintained properly, a buccal swab can easily be contaminated with bacterias. Further, the yield of DNA is less using a buccal swab.

Blood is one of the common and most routinely used biological samples in DNA extraction.

DNA extraction:

We have to first obtain DNA.

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.

You can use either of DNA extraction method enlisted below,

  1. Phenol-chloroform DNA extraction method
  2. CTAB DNA extraction method
  3. Proteinase K DNA extraction method

However, we strongly recommend using ready to use DNA extraction kit for DNA fingerprinting.

For the DNA test, the purity of DNA must be ~1.80 and the quantity around 100ng. Purify the DNA using the purification kit, if needed.

After that, quantify the DNA using the UV-Visible spectrophotometer. And perform one of the following method listed below,

DNA fingerprinting techniques:

Three common methods are used: 

  1. RFLP based STR analysis
  2. PCR based analysis 
  3. Real-time PCR analysis

1. RFLP based STR analysis:

Restriction fragment length polymorphism was the first method used in the analysis of DNA fingerprinting.

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 southern blot analysis is performed

Chemical denaturation is performed to fix the DNA on nitrocellulose paper.

hybridization is performed using the radio-labelled probes. It finds its complementary sequence and binds on 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. Non-bounded probes create background noise in autoradiography.

Now our hybridized blot is ready. It is exposed to X-ray film for analysis of results. Different DNA bands are observed as per probe-hybridization.

The present method is widely used for DNA fingerprinting, nonetheless, this technique has several limitations. 

The method is tedious, takes a lot of time and hence it is expensive. Also, a large amount of DNA required. Therefore, it is not recommended for forensic analysis.

2.  PCR based analysis:

The present method is one of the most popular methods of DNA fingerprinting.

It is simpler than RFLP-autoradiography and gives accurate results than the traditional techniques.

PCR based DNA fingerprinting is 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. Using polymerase chain reaction, millions of copies of DNA are obtained.

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. Based on the size of DNA fragments, different DNA bands are observed in a gel.

However, PCR based gel electrophoresis is mostly used for VNTR analysis. VNTRs are larger than STRs hence it separates properly.

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.

The image represents the general process of DNA fingerprinting

Nowadays, a state of the art method called capillary gel electrophoresis which can separate minute DNA fragments, precisely.

Capillary gel electrophoresis gives the best result for both STR and VNTR because it can discriminate even nearest DNA fragments.

In the present method, we can identify the fragments of STR or VNTR but we can’t count the exact number of the repeats of a suspect.

3. Real-time PCR analysis:

Real-time PCR is able to amplify DNA as well as counts the number of amplicons.

Therefore the amount of the repeats present in a sample can be quantified.

The process is quick, reliable and accurate therefore it is widely adopted in criminal identification.

Furthermore, it can amplify DNA from a small amount of sample.

In RT PCR fluoro labelled probes are used, Instead of simple complementary 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 for different repeats. Here separate gel electrophoresis doesn’t require therefore this technique is so fast and very reliable.

We can calculate the number of repeats present into different samples.

In addition to this, using DNA sequencing, a sequence variations like SNP or other mutation can also be determined. In forensic analysis, RT-PCR with the capillary gel electrophoresis is used for accurate quantification of repeat numbers.

Applications of DNA fingerprinting:

Using the DNA fingerprinting method, the biological identity of a person can be revealed. 

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 also verified using DNA fingerprinting.

Badly damaged dead bodies can also be identified. 

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, sometimes.

Contamination increases the chance of false-positive results, especially in the case of carrier identification.  

Using VNTRs and STRs markers with PCR-gel electrophoresis, maternal cell contamination can identify during the pregnancy genetic testing. 

DNA fingerprinting

The image represents the maternal cell contamination by VNTR

The image represents the maternal cell contamination by VNTR

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 crime site, could be saliva, blood, hair follicle or semen.

DNA is extracted and analysed against the suspect, using the two markers we explained above. 

 Different countries have different criteria for the use of STR. 13, 11 and 12 STRs are commonly used in criminal verification in the USA, UK and India, respectively. 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.

Besides these, the present method is often employed for checking graft rejection in case of organ transplantation. Known as HLA typing, different markers of HLA region genes are amplified and matched between donor and recipient. 

The exact match score indicates graft acceptance. This means an organ can be transplanted. 

DNA testing or fingerprinting is also used for identification of inherited and non-inherited genetic disorders, besides the above applications. 

My experience of DNA fingerprinting

During my tenure of prenatal research, instead of following the standard protocol with STR or VNTR, I had prepared our own combination of markers.

I had selected 4 VNTRs, 3 STR, 1 mitochondrial DNA marker and 1 Y chromosome-specific STR for detection of maternal cell contamination.

Mitochondrial DNA is additionally a great tool for verifying individuals. As different cells have different numbers of mitochondria, the number of mitochondrial DNA also varies from individuals to individual.

Y specific marker helps in gender verification. This one is alway included in forensic analysis. 

Conclusion:

The DNA fingerprinting method is an unmatched technique since long, although, modifications are done to increase the sensitivity and precision. Still, restriction digestion and PCR amplification are still the best and reliable option for profiling DNA.