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DNA fingerprinting: Steps and Applications, Practice Problems and FAQ’s

 DNA fingerprinting: Steps and Applications, Practice Problems and FAQ’s

How many of you enjoy watching crime thrillers and murder mysteries? My favourite part of a murder mystery is the forensic analysis of the different biological samples that most accurately point towards the actual murderer. You must have seen forensic officials use blood, hair, dead cells, saliva, etc found from a crime scene to solve mysteries. These biological samples can be used to extract DNA and the unique DNA sequence of the sample can be used to identify a suspect. In fact, in a crime investigation in a Finnish country in 2008, blood drawn out from a dead mosquito found in the crime scene was used by a to extract DNA, sequence it and finally find the felon.

You must be wondering how DNA can be used to identify a person. Well there are approximately 7.9 billion human beings on Earth and although we have differences in our race, creed, ancestry, appearances and personalities, around 99.9% of the genetic makeup of all human beings is identical. The variations lie in 0.1% of the genome and this 0.1% of the genome is very unique to every individual. The unique nucleotide sequences of this 0.1% of the human genome, which varies from one person to another, can be compared to the unique fingerprint that each human being has and can be used to specifically identify a human being. The technique used to identify such unique sequences is called DNA fingerprinting and that is what we are going to discuss in this article.

Table of contents:

  • DNA fingerprinting
  • Steps involved in DNA fingerprinting
  • Applications of DNA fingerprinting
  • Practice problems
  • Frequently Asked Questions

DNA fingerprinting

DNA fingerprinting is a technique which is used to determine the nucleotide sequences of certain areas of DNA which are unique to each individual. It is used to compare the DNA of two individuals. The technique of DNA fingerprinting was first discovered by Sir Alec Jeffreys.

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Like we have already mentioned, around 99.9% of the human genome is identical in all humans but 0.1% of the genome varies from one one human being to another and is unique to each one of us. This creates the premise of DNA fingerprinting and using these unique sequences as a mean to identify individuals. But, before we go into the details of how DNA fingerprinting is done, let us first understand what is this unique 0.1% of the genome composed of and how does it arise?

Repetitive DNA

0.1% of the human genome shows differences in the base sequences and these differences make every individual unique. The differences not only occur in genes but also in repetitive DNA or satellite DNA. Repetitive DNA sequences are a part of introns or non-coding sequences of our genome that are repeated several times throughout our genome.

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The number of repeats or copy numbers varies from person to person. In an individual, the number of copies in different chromosomes is also different, but the copy number in the overall genome will be constant for all cells in an individual. For example, person 1 shows five repeats, whereas person 2 shows seven repeats.

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If we perform density gradient centrifugation of human DNA, two different bands are obtained on the basis of density. These are a lighter band on top which is less dense and a heavier band at the bottom with more density.

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The amount of DNA present in each of these bands can be measured by extracting the DNA in the bands and measuring their absorbance through spectrometry. A graph plotted between the amount of DNA in the bands and their density gives us two peaks. The major peak is obtained from the bulk DNA which also shows greater density and forms the heavy band at the bottom of the centrifuge tube. A minor peak is formed by the repetitive DNA or satellite DNA which is lesser in amount and density and hence forms the light band at the top of the centrifuge tube. The words ‘satellite’ is used to describe these DNAs because it comes from the word ‘follower’ and the minor peak formed by these repetitive DNA sequences follow the major peak formed by the bulk DNA.

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Types of Satellite DNA

Satellite DNA is classified into two types on the basis of three factors. These factors are:

  • Length of a sequence
  • Number of repetitive units
  • Base composition i.e. A:T/G:C composition

The two types of satellite DNA are microsatellite and minisatellite.

Microsatellites

Microsatellite is a region of repetitive DNA that has a length of 2 to 6 base pairs. Most of the microsatellites are found in the non-coding region of a DNA. If they are present in the coding region, this can lead to diseases and phenotypical changes. Microsatellites are also known as short tandem repeats (STRs). These sequences are used in DNA fingerprinting.

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Minisatellites

Minisatellite is a region of repetitive DNA that has a length of 10-100 base pairs. Minisatellites are also known as a variable number of tandem repeats (VNTRs). They are found in the telomeres and centromeres of chromosomes. These repetitive sequences do not code for any protein. They are most commonly used in DNA fingerprinting because these sequences show a very high degree of variation.

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Here, the tandem stands for ‘one after the other’. If there is a break in between, this is called non-tandem repeats.

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Mutation

Mutation is defined as the alteration in the DNA sequence that can occur due to error in DNA replication at the time of cell division or may be induced due to exposure to radiation or other mutagens. Mutations may result from addition of nucleotide, deletion of nucleotide or conversion of a purine base to another purine base or pyrimidine base or vice versa.

If these mutations occur in repetitive sequences then, the mutations can change the total number of repetitive sequences. Somatic mutations occurring in the somatic cells are not inherited but the germinal mutations in the germ cells are inherited by the offspring.

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Satellite DNA does not code for any protein. Therefore, the mutations that occur in satellite DNA do not result in genetic disorder. The changes occur due to mutation accumulating in every generation and this leads to DNA polymorphism.

DNA polymorphism

DNA polymorphism is a phenomenon in which DNA exhibits differences in sequences among the individuals of a population. Polymorphism occurs when a mutant at a locus exists with a frequency greater than 0.01 in a human population. The phenomenon of polymorphism plays a significant role in evolution and speciation. There are two types of polymorphism. These are single nucleotide polymorphism and multiple nucleotide polymorphism.

Single nucleotide polymorphism

The single nucleotide polymorphism occurs only in one nucleotide. This will change the copy number of repeats.

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Multiple nucleotide polymorphism

Multiple nucleotide polymorphism occurs in many nucleotides. This also leads to a change in copy number of repeats.

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Steps involved in DNA fingerprinting

There are six steps involved in the process of DNA fingerprinting. These are listed below:

  • DNA isolation
  • Restriction digestion
  • Electrophoresis
  • Southern blot
  • Hybridisation
  • Autoradiography

DNA isolation

DNA can be isolated from any biological sample, such as hair follicle, saliva, blood, skin cells or the semen. The biological sample can be referred to as any living cell of a person.

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Restriction digestion

Firstly, if the amount of sample is too low, amplify it using Polymerase Chain Reaction. This will give multiple copies of a sample which is then used for restriction digestion. In this process, fragments are made using restriction endonuclease enzymes or molecular scissors. Restriction endonuclease cuts the DNA into multiple fragments and therefore, this process is known as restriction digestion.

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Electrophoresis

The DNA fragments that are digested with restriction endonuclease enzymes are added into the electrophoresis unit. Electrophoresis is a technique used to separate macromolecules on the basis of size to charge ratio. The DNA is negatively charged and therefore, it is added to the negative electrode. Under the influence of the electric field, the DNA molecules move towards the positive terminal.

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Southern blot

Southern blotting is a technique in which the separated DNA fragments are transferred to the synthetic membrane. This synthetic membrane is a nitrocellulose membrane or nylon membrane. The separated fragments of DNA are attached to the synthetic membrane.

Hybridisation

For the process of hybridisation, we need to design a probe for the VNTR we are looking for. This radio-labelled probe or a short DNA sequence that is complementary to the desired DNA fragment or VNTR is introduced into the membrane. The probe attaches to the VNTRs present on the membrane. The probes that are unable to attach are washed away.

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Autoradiography

Autoradiography is a technique used to visualise the fragments of DNA molecules that are radioactively labelled. The nylon membrane in which probes are attached to the VNTRs is exposed to X-Ray that show radiolabeled VNTRs.

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Applications of DNA fingerprinting

The technique of DNA fingerprinting is used in the following situations:

  • Paternity and maternity testing: DNA polymorphisms are inherited from parents to offspring. Therefore, they are used in solving parental disputes.
  • Criminal identification and forensics: In an individual, all the cells have the same degree of polymorphisms. Therefore, this is used in locating suspects and culprits.
  • Population and genetic diversities: DNA fingerprinting is used to determine population and genetic diversities on the basis of repetitive sequences.

Practice Problems

1. From the given options, identify the DNA fingerprint autoradiogram of which two individuals are not different?

a. Dizygotic twins
b. Monozygotic twins
c. Siblings
d. Mother and child

Solution: DNA fingerprinting is a laboratory technique for identifying people based on their DNA's distinctive repeated sequence. Due to differences in their genetic makeup, two people's DNA fingerprints are distinct. Because of the large number of these variants, each person's DNA sequence is unique. Monozygotic twins develop from the same zygote. As a result, they are genetically identical. Therefore, restriction digestion will produce similar DNA fragments and their DNA fingerprint autoradiogram will be the same. Hence, the correct option is b.

2. Identify the incorrect statement regarding satellite DNA.

1. Satellite DNA always code for proteins
2. Satellite DNA shows a high degree of polymorphism
3. Satellite DNA are repetitive

a. Only I
b. I , III
c. I, II
d. II, III

Solution: Satellite DNA are highly repetitive DNA sequences that do not code for proteins and are used for DNA fingerprinting. Satellite DNA is classified into two types on the basis of three factors. These factors are:

  • Length of a sequence
  • Number of repetitive units
  • Base composition i.e. A:T/G:C composition

The two types of satellite DNA are microsatellite and minisatellite. Hence, the correct option is a.

3. Determine which is not the requirement of DNA fingerprinting?

a. Southern blotting
b. DNA-RNA hybridisation
c. Restriction enzymes
d. DNA-DNA hybridisation

Solution: DNA fingerprinting is a laboratory technique for identifying people based on their DNA's distinctive repeated sequence. Due to differences in their genetic makeup, two people's DNA fingerprints are distinct. Because of the large number of these variants, each person's DNA sequence is unique except for the monozygotic twins. The DNA fingerprinting involves the following steps:

  • DNA isolation
  • Restriction digestion
  • Electrophoresis
  • Southern blot
  • Hybridisation
  • Autoradiography

Therefore, it does not involve DNA-RNA hybridisation. Hence, the correct option is b.

4. From the given options, determine which is not an application of DNA fingerprinting?

a. Forensic studies
b. mRNA sequencing
c. Paternity testing
d. Determining population diversities

Solution: DNA fingerprinting is a laboratory technique used for identifying people based on their DNA's distinctive repeated sequence. The DNA fingerprint of two people is distinct due to differences in their genetic makeup. The technique of DNA fingerprinting has the following applications:

  • Paternity and maternity testing: DNA polymorphisms are inherited from parents to offspring. Therefore, they are used in solving parental disputes.
  • Criminal identification and forensics: In an individual, all the cells have the same degree of polymorphisms. Therefore, this is used in locating suspects and culprits.
  • Population and genetic diversities: DNA fingerprinting is used to determine population and genetic diversities on the basis of repetitive sequences.

On the other hand, mRNA sequencing is a technique for determining the nucleotide sequence of messenger RNA. The sequences contained in mRNA cannot be determined by DNA fingerprinting. Hence, the correct option is b.

FAQs

1. How is DNA fingerprinting different from DNA profiling?
Answer:
Although the DNA fingerprinting and DNA profiling are used synonymously nowadays, there lies a thin line of difference between the two. The original DNA fingerprinting method introduced by Sir Alec Jeffreys focused on detecting variations in the form of minisatellite patterns in the genome which is unique to every individual. Thus, it helps in identification of individuals. DNA profiling is a modern technique of determining the DNA characteristics of an individual using short tandem repeats (STRs) or microsatellites.

2. What are the limitations of DNA fingerprinting?
Answer:
There are several limitations to the process of DNA fingerprinting. Firstly, the process is complicated, tedious and it requires expertise to interpret the results. Incorrect interpretation of the results can have grave consequences. Also, DNA samples are fragile and can get easily contaminated, making the test results inaccurate. To avoid such phallacies, often tests have to be run a number of times on multiple samples to get accurate results. The data collected from DNA profiling needs to be properly stored and protected as this data can be gravely misused if it reaches the wrong hands.

3. When was DNA fingerprinting first used?
Answer:
DNA fingerprinting was first used in the 1980s in Britain to identify a boy as the son of an English woman to protect him from deportation in an immigration case.

4. How widely is evidence obtained from DNA profiling accepted in the criminal justice system?
Answer:
Although the concept of DNA fingerprints, variations in minisatellite patterns and the process of DNA fingerprinting is not necessarily understood by majority in the public, still evidence obtained against a suspect from DNA profiling appears to be widely accepted by all. All state courts and federal courts accept such evidence to be legit.

YOUTUBE VIDEO: https://youtu.be/TrdmXnqnrh4 

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