“A laboratory genetic technique or method to identify individuals using bodily samples such as blood, saliva, or hair is referred to as DNA fingerprinting.”

Or we can define DNA fingerprinting as, “A DNA test to establish a link or relation between two person or living organisms by analyzing their STR and VNTRs is known as DNA fingerprinting.”

DNA profiling, DNA testing, DNA analysis, Genetic profile, DNA identification, genetic fingerprinting, and genetic analysis are some of the popular names used for DNA fingerprinting.

The present method is employed usually in criminal verification and crime scene investigation. However, it is also applicable to establishing a relationship between two persons and to know someone’s identity. The testing method is practiced not only for humans but also for any organisms present on earth.

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

DNA is our blueprint, basis of life, encodes proteins, and regulates gene expression. It is made up of sugar, phosphate, and nitrogenous bases. DNAs are located on chromosomes. The whole set of DNA or chromosomes is known as the genome. Interestingly, there are several regions in our genome that are unique and hypervariable.

Every person or organism has different patterns for those regions using which a DNA fingerprinting is performed.

In the present article, we are going to talk about DNA fingerprinting and how it is done. We will also talk about various techniques and methods to do so, scientifically. So let’s start the story,

What is DNA fingerprinting?

The technique of DNA fingerprinting changed the era of identification, characterization, and classification of organisms.

Notably, approximately 99% of our DNA (deoxyribose nucleic acid) is similar. A 0.1% difference is sufficient to make someone so unique. We are using this 0.1% portion for DNA profiling which is often known as DNA fingerprinting.

There is a reason, why only DNA is used,

In all bodily parts, tissues, and cells (except germ cells) our DNA is the same. Even, after the death of the individual, it remains the same. It can remain stable even after 1000 years.

As we know, 97% of our genome is non-coding, repetitive, and junk, protein formation is regulated by only 3% portion, we know it as genes.

Moreover, repetitive nature and polymorphic properties of junk DNA are utilized to perform DNA fingerprinting. The number of repeats and the sequence structure of those regions varies between individuals and organisms based on that the entire DNA profile or the DNA print can be prepared.

A comprehensive overview of the whole method is given below,

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

Though the PCR based method for DNA fingerprinting is accurate and too fast but after the discovery of real-time PCR and DNA sequencing, it becomes even more powerful. The molecular tools available nowadays create 100% accurate patterns of a person’s DNA.

Definition:

“Using laboratory techniques such as PCR, DNA sequencing or restriction digestion, a unique pattern of a person or any living thing can be prepared using DNA fingerprinting.”

History:

Since the discovery of DNA in 1953 by Watson and Crick, genetic science has become more advanced and accurate. To know more on DNA and history of genetics, please read this article: DNA: Definition, Structure, Function, Evidence, and Types

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. His method was actually a combination of 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 and abnormal repeats halt DNA replication. It clearly indicates that satellites help to do proper replication. Mutation in those sequences causes the end replication errors or problems.

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

Mainly two types of satellite regions are present in the 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.

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

Contrary, microsatellites are smaller than minisatellites. It’s 1-6 bp long and repeated 5 to 10 times in a genome. For example, STR and SSR. Read more on STR- short tandem repeats.

These regions are also hypervariable, non-coding and telomeric, likewise the VNTRs.

We have covered the entire article on VNTR and STR in genetic marker, for a more detailed 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 assumes that the name satellite is given because the sequences are located on the telomeric region of chromosomes but this conception is not true.

Because of their nature of separation in the centrifugation process, the name satellite DNA is given.

A larger 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 coworkers had identified the first microsatellite. The DNA fingerprinting was performed through RFLP & autoradiography by them.

Jeffrey had performed restriction digestion using REase and separated various DNA fragments using agarose gel electrophoresis. In the next step, the separated DNA fragments were transferred to a nylon sheet to perform southern hybridization. Radio-labeled probes were hybridized to detect various fragments.

The results were analyzed with the help of the X-ray film. This was the first method adopted by scientists to prepare a DNA fingerprint.

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:

Gene exists in nature because it can encode various proteins. Through the collective efforts of replication, transcription and translation, DNA, mRNA, and proteins form, respectively. But the DNA other than genes aren’t able to construct any protein, however, as per recent findings, non-coding DNA regulates gene expression.

Cell, tissue, and organs specific gene expression is regulated by the loosely packed, junk, and non-coding DNA. Read more: Gene regulation.

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 this specific VNTR and STR in another individual is almost negligible.

Nonetheless, in the case of monozygotic twins, it is quite possible.

Monozygotic twins are developed from a single type of embryo (due to the splitting of an embryo), so the chances of having the same VNTR and STR profile are higher.

Still, monozygotic twins aren’t similar biologically. Read this article to know more: 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

Here we have explained one simple example to understand the topic more precisely. Various VNTRs (V1, V2, V3, V4 and V5) are digested with a single type of restriction endonuclease, and the fragments are serrated on the agarose gel. The results are shown in the figure below,

Now, let’s understand the actual process.

Steps:

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

Process of DNA fingerprinting:

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

Step 1: Sample collection:

DNA can be obtained 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 samples used.

In criminal cases, a buccal swab is taken usually. The buccal swab sample collection method is non-invasive and handy.

However, if not maintained properly, a buccal swab can easily be contaminated with bacteria. Further, the Buccal swab DNA yield is very less. A blood sample is a good replacement for a buccal swab sample. We can use a blood sample as well.

Step 2: DNA extraction

We have to first obtain DNA.

To perform any genetic applications, DNA extraction is one of the most significant steps. Good quality and quantity DNA increases the possibilities of getting good results.

You can use either of DNA extraction method enlisted below,

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

The purity and quantity of DNA should be ~1.80 and 100ng, respectively to perform the DNA test. Purify the DNA using the DNA 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:

Step 3: Restriction digestion, amplification or DNA sequencing

Three common methods are used:

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 for DNA fingerprinting. Here, the restriction endonuclease- a type of nuclease cleaves DNA in various sized fragments that are separated on a gel usually an agarose gel.

Each DNA fragment runs at different speeds due to size differences. Now the fragments are transferred to the nitrocellulose paper to perform the Southern blotting. To fix DNA on a nitrocellulose paper, chemical denaturation is performed.

In the next step, probe hybridization occurs using the radiolabeled probes. The probes find its complementary sequence and bind to it.

The probes and denatured DNA on nitrocellulose paper are now incubated for several hours to hybridize. Every time when a probe finds its complementary sequence it will bind to it. Using a washing step, unbounded probes are removed. The unbounded probes complicate our results.

Now our hybridized blot is ready. It is exposed to an X-ray to get results. Different DNA bands are observed in X-ray films. The present method is widely used for DNA fingerprinting, nonetheless, it has several limitations.

The method is tedious- takes so much time, and hence more expensive. Also, a large amount of DNA is required. Therefore, it is not recommended to analyze forensic samples.

What to know the difference between probe and primer? Read this article: Difference Between Probe vs Primer.

2. PCR based analysis:

The PCR based DNA fingerprinting technique is most popular among all. It is simpler than RFLP-autoradiography and gives reliable results than traditional techniques.

PCR based DNA fingerprinting is faster and more accurate. The brief overview is given into the figure below,

Now we have numbers of known STRs and VNTRs present in the NCBI database. We can choose any of the STR or VNTR of our interest. After choosing the sequence, primers are designed based on the sequence data.

The primer set is used to amplify the DNA region during PCR. But first, DNA extraction is performed to get copies of DNA to in polymerase chain reaction. Simple agarose gel electrophoresis set up is enough to obtain results.

Here, the amplified DNA fragments are analyzed on the agarose gel. Based on the size of DNA fragments, different DNA bands appear in a gel. However, PCR based gel electrophoresis is mostly used for VNTR analysis. Because VNTRs are larger than STRs hence it separates properly.

STRs are smaller in length so it is difficult to distinguish them in an agarose gel. In contrast, VNTRs are larger fragments and give a beautiful distinct band pattern in agarose gel electrophoresis.

Nowadays, recently evolved state of the art method known as capillary electrophoresis has the potential to separate even minute DNA fragments, precisely. It can discriminate nearer fragments of single basepair difference thus for both VNTR and STR analysis, the present method is employed so often.

However, we can’t count the number of repeats present in a DNA sequence using the capillary gel electrophoresis.

3. Real-time PCR analysis:

Real-time PCR is able to amplify DNA as well as counts the number of amplicons. Therefore the number of repeats present in a sample can be calculated.

The Real-Time PCR method is widely used in crime scene investigation and criminal verification due to its power to quantify the DNA present in a sample. It is quick, accurate, reliable, and cheaper than other methods. Moreover, it can even quantify a smaller amount of DNA from any sample.

In RT-PCR, fluoro-labeled probes are used, Instead of simple complementary primers. However, the probes are not so long as it is used in the southern blot.

Once the fluoro-labeled probe is bound to its complementary sequence, it will give a signal to the machine which monitors the amplification process of that particular fragment.

Based on the fluorescent signals obtained, it creates different peaks for different repeats. Here, gel electrophoresis doesn’t require therefore this technique is so fast and very reliable.

We can calculate the number of repeats present in different samples. A pictorial representation of three different DNA bands of mother, child, and father is shown in a figure below,

In addition to this, using DNA sequencing, sequence variations like SNP (Single Nucleotide Polymorphism) or other mutations can also be determined. In forensic analysis, RT-PCR with the capillary gel electrophoresis is used for accurate calculation of repeat numbers and separation of different fragments.

Step 4: Analysis of results

As we discussed, using the southern blotting, agarose gel electrophoresis, capillary electrophoresis, real-time amplification, and DNA sequencing, the results for various DNA profiling can be obtained in which rt-PCR and sequencing are frequently used in forensic.

Step 5: Interpreting results

By comparing DNA profiles of various samples, variations and similarities between individuals can be identified. Notably, the entire process is now almost automated. We do not have to do anything, the computer gives us the final results.

Applications of DNA fingerprinting:

Using the DNA fingerprinting method, the biological identity of a person can be revealed. For validating one’s identity, there is no other better option than DNA fingerprinting.

Badly damaged dead bodies can be identified.

It is used to detect 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 be identified during pregnancy genetic testing.

One of the most important applications of the present technique is in the crime scene investigation and criminal verification.

The sample is collected from the crime site which could be saliva, blood, hair follicle, or semen. DNA is extracted and analyzed against the suspect, using the two markers we explained above. By matching DNA band patterns criminal’s link to crime can be established.

Different countries have different criteria to use STRs. 13, 11 and 12 STRs are commonly used in criminal verification in the USA, UK, and India, respectively. As the number of STR markers increases the accuracy of the result also increases.

Identification of blood relatives:

No two individuals are genetically identical. To establish or to know blood relation between two unrelated individuals, the present method is adopted.

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.

Moreover, scientists use the present method to screen inherited & non-inherited disease, to find genetic abnormalities, to detect any genetic disorders or mutations, and to create phylogeny between various organisms.

My experience of DNA fingerprinting

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

I had selected 4 VNTRs, 3 STR, 1 mitochondrial DNA marker and 1 Y chromosome-specific STR for the 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 among individuals. Y specific marker helps in gender verification. This one is always included in forensic analysis.

Conclusion:

Since long, scientists are using DNA fingerprinting for various applications. Notwithstanding, changes kept occurring from time to time, to make the technique more accurate. PCR is a powerful tool. It has given accuracy, sensitivity, precision, and low-cost assay to DNA fingerprinting.

Still, restriction digestion is one of the most popular assays utilized to teach students genetic testing.