The Peculiarities Of DNA Fingerprinting

ABSTRACT

DNA Fingerprint is a real technique that is used in labs. DNA fingerprint was discover in 20th Century. This survey quickly thirty years of progress in measurable. DNA sample that accrued in crime scene like stains of blood, semen, saliva or hair born material as well.

In DNA Fingerprint many applications are used for many different purpose which contain advantages and disadvantages too. Now a day it is very useful technique.

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INTRODUCTION

What is DNA Fingerprint?

DNA finger print was invented in 1984 by “Sir Alee Jaffrey’s” onward, he would realized that detection of human variation.

The technique of DNA fingerprint is commonly detects lots of “minisatellite” that are present in genome for the production of unique pattern in a single person or individual. This process is known as DNA fingerprint. The first minisatelites was discovered in 1980. This technology fingerprinting has experienced many changes with many possibilities occurring DNA fingerprinting and the current technology used in DNA process.

What is meaning of the MINISATELLITES

A minisatellite is a tract of repetitive DNA in which certain DNA motifs (ranging in length from 10–60 base pairs) are typically repeated 5-50 times. Minisatellites occur at more than 1,000 locations in the human genome and they are notable for their high mutation rate and high diversity in the population. Minisatellites are prominent in the centromeres and telomeres of chromosomes, the latter protecting the chromosomes from damage. The name 'satellite' refers to the early observation that centrifugation of genomic DNA in a test tube separates a prominent layer of bulk DNA from accompanying 'satellite' layers of repetitive DNA. Minisatellites are small sequences of DNA that do not encode proteins but appear throughout the genome hundreds of times, with many repeated copies lying next to each other.

DISCOVERY

The first human minisatellite was discovered in 1980 by A.R. Wyman and R. White, Discovering their high level of variability, Sir Alec Jeffrey’s developed DNA fingerprinting based on minisatellites, solving the first immigration case by DNA in 1985, and the first forensic murder case, the Ender by murders in the United Kingdom, in 1986. Minisatellites were subsequently also used for genetic markers in linkage analysis and population studies, but were soon replaced by microsatellite profiling in the 1990s.

The term satellite DNA originates from the observation in the 1960s of a fraction of sheared DNA that showed a distinct buoyant density, detectable as a ‘satellite peak’ in density gradient centrifugation, and that was subsequently identified as large centromere tandem repeats. When shorter (10–30-bp) tandem repeats were later identified, they came to be known as minisatellites. Finally, with the discovery of tandem iterations of simple sequence motifs, the term microsatellites was coined.

The first human minisatellite was discovered in 1980 by A.R. Wyman and R. White, Discovering their high level of variability Sir Alec Jeffrey’s developed DNA fingerprinting based on minisatellites, solving the first immigration case by DNA in 1985, and the first forensic murder case, the Ender by murders in the United Kingdom, in 1986. Minisatellites were subsequently also used for genetic markers in linkage analysis and population studies, but were soon replaced by microsatellite profiling in the 1990s.

The term satellite DNA originates from the observation in the 1960s of a fraction of sheared DNA that showed a distinct buoyant density, detectable as a ‘satellite peak’ in density gradient centrifugation, and that was subsequently identified as large centromeric tandem repeats. When shorter (10–30-bp) tandem repeats were later identified, they came to be known as minisatellites. Finally, with the discovery of tandem iterations of simple sequence motifs, the term microsatellites was coined.

METHOD OF DNA FINGERPRINTING

Six steps to understanding DNA fingerprinting:

• Extracting the DNA from cells
• Cutting up the DNA using an enzyme
• Separating the DNA fragments on a gel
• Transferring the DNA onto paper
• Adding the radioactive probe
• Setting up the X-ray film

Extraction of DNA

DNA can be thought of as a length of letters A, C, G and T (6 billion of them in a human cell). Some letters code for proteins, which then do stuff in the cell (like making the cell move or speeding up chemical reactions). Other letters do nothing at all and are just “spacer” or “junk” DNA. The DNA is a double helix, with the letters facing each other and paired up, so that “A” matches with “T” on the other strand, and “C” matches with “G” on the other strand. The first step in DNA fingerprinting is getting your DNA in a pure form. You can get DNA from any cell/tissue such as muscle, semen, saliva but blood is normally easiest. The blood is treated with a series of chemicals until pure DNA emerges as a white solid. The DNA is stored, dissolved in essentially water, in a small plastic tube and kept in a fridge until ready for the next stage.

Cutting up the DNA

Freshly extracted DNA in water is quite sticky, because the DNA strands are very long. They are too long to be separated in the gel in the next stage. The next step is to cut up the DNA strands using a “restriction enzyme”. This “restriction enzyme” doesn’t cut randomly in the DNA, but at specific letter sequences. This stage involves adding the restriction enzyme (colourless liquid) to the DNA (another colourless liquid), using a pipette. The enzyme takes a few hours to cut at all the places it can in the DNA strands.

Separating the DNA fragments on a gel

The gel is like a sieve, in that it separates the different sizes of DNA fragment generated by cutting up the DNA. We add a blue dye to the DNA fragments using a pipette, and use a pipette to move the blue DNA liquid from a colourless tube into the “well” – little hole – in the gel (see the top picture on the next page). We use a blue dye to see where we have added the DNA on the gel – it’s just for our benefit so we don’t add two different DNA samples in the same hole! There would be a DNA sample from several people, each sample in a different hole.

The gel is made from something called agarose (derived from seaweed) and is just a pure firm jelly. The gel is placed in a colourless liquid and electrodes are attached to the gel equipment, and a power supply is turned on. By putting the liquid DNA fragments in the hole at one end and passing an electric current through the gel, the DNA fragments move into the gel with the electric current. Small fragments move faster than larger fragments, so the DNA fragments are separated as they move in the gel. After several hours the gel is ready. The gel is checked by shining ultraviolet light on it to check for a nice strong DNA smear (see bottom picture on the next page).

In the gel there is a chemical called “ethidium bromide” which sticks to all DNA fragments and allows the DNA to be seen when an ultraviolet light is shone on it. At this stage, the DNA can be seen as a smear in the gel rather than the “lots of bands” that is characteristic of DNA fingerprints – that is what comes later.

A gel (similar to one Alec would have used, for DNA fingerprinting, they are larger but essentially the same). Notice the holes on the right where the DNA is added. The DNA would move right to left when an electric current is applied.

What separated DNA fragments look like under ultraviolet light – a smear. Picture is orientated like the picture above – the holes are by the labels. Colour here is artificial – DNA is normally pink under ultraviolet light.

Transferring the DNA onto paper

The gel-separated DNA fragments (the smear shown above) are converted to single stranded fragments by dunking the gel in weak acid, so that the DNA letters are exposed, rather than being in the middle of the double helix. The gel-separated DNA fragments are then transferred to white nitrocellulose paper, so the paper now carries an exact replica of the DNA on the gel. This is called “Southern blotting”. The Southern blot equipment is quite Heath Robinson, involving trays, paper towels, and lots of solutions so can get quite messy. Heavy books are placed on top of the towels to squash everything down. The blot can be left overnight, typically.

Adding the radioactive probe

This is the clever bit. Most of the method I have described is quite routine in the lab since the late 1970s and not developed by Alec but by Ed Southern at Oxford (hence “Southern blotting”). Alec’s particular contribution was the choice of the “probe”. This “probe” determines which DNA fragments can be seen at the end of experiment. It is a small chunk of radioactive DNA of a particular sequence of letters. The probe sticks to the fragments of the DNA that has the matching sequence, but only those fragments that have the matching sequence of letters, no other fragments.

In DNA fingerprinting the probe is a sequence of 33 letters that is found in the repeated “stutters” of the genome. Therefore, only the DNA fragments that contain these repeated “stutters” are seen at the end of the experiment. They are seen as the dark bands you will be familiar with, on a DNA fingerprint.

Essentially, to put it another way, there are lots and lots of differently sized DNA fragments on the nitrocellulose paper (remember the smear from the gel). What we have done is “ask the paper” which fragments have a particular sequence of letters within them. Those are the ones that appear as dark bands.

The nitrocellulose paper and the probe (colourless, radioactive liquid) are placed together in a glass tube in a hybridisation oven at 65 degrees Celsius(think a rotisserie) for an hour or two, so that the probe covers the paper and can stick to the DNA fragments with the matching sequence. The nitrocellulose paper is then rinsed to remove any radioactive probe liquid that has not stuck. The paper should be mildly radioactive because of the probe stuck to it – it should make a nice crackling noise (not screaming, not silent) when the Geiger counter detector is passed over the paper. All of this stage is done in a working area set aside for radioactivity.

Setting up the X-ray film

In the dark room, the nitrocellulose paper is placed against a piece of X-ray film, in a large film cassette (typically bigger than A3 size). The X-ray film can record the pattern of radioactivity on the paper – i.e. where the probe has stuck. Therefore the X-ray film, when developed, will have the pattern of bands which are the DNA fragments where the probe has stuck. The film cassette is shut and your name and date written on a bit of masking tape on the outside. It’s left on the bench overnight, or over the weekend, so that the film is exposed to the radioactivity for long enough to make an image.

ADVANTAGES AND DISADVANTAGES OF DNA FINGERPRINTING

Advantages of DNA Fingerprinting

• it is an easy and painless method for the subject being tested. It is less invasive then taking a blood sample
• It is an affordable and reliable technique
• It can be conducted in a relatively short amount of time
• Anyone at any age can be tested with this method without any major concerns
• There is a large variety of uses such as in legal claims, missing person’s cases, identification for the military, and paternity and prenatal testing
• The technique has used since 1984, making it highly developed and improved.

Disadvantages of DNA Fingerprinting

• the sample of DNA can easily be ruined during the process of DNA fingerprinting, causing the sample to become completely useless for testing
• The process itself is complex and tedious, and can give results that may be hard to interpret
• The test needs to be run on multiple samples, a numerous amount of times for ideal accuracy. Commonly, labs run each test twice with four samples.
• Privacy issues could occur if the information isn't kept secure at the lab. Personal information legally can only be released with a written order. This personal information if leaked, could potentially complicate insurance processes, health care and job prospects for an individual

REFRENCES

1. https://www2.le.ac.uk/offices/press/for-journalists/code-of-a-killer-1/a-beginners-guide-to-dna-fingerprinting
2. https://allaboutdnafingerprinting.weebly.com/advantages-and-disadvantages.html
3. https://www.yourgenome.org/facts/what-is-a-dna-fingerprint?fbclid=IwAR3XwbKSwJsCIms6xheK9DINadaw1I-8zGzzUYaf6YEyUM2b5gx6cmNqsCg
4. https://www.infoplease.com/encyclopedia/science/biology/genetics/dna-fingerprinting/methods?fbclid=IwAR00F7uvjuAU9ZeK5K9_o5aPXjv1XF4U4bZW6hekaQbAw3PW5rNriKLxDQ
5. https://dalspace.library.dal.ca/bitstream/handle/10222/77851/VOLUME%2031-NUMBERS%201-3-2001-PAGE%20213.pdf?sequence=1
6. https://www2.le.ac.uk/offices/press/for-journalists/code-of-a-killer-1/a-beginners-guide-to-dna-fingerprinting?fbclid=IwAR0eYY6tmQThNHERR3Q5nDcDahquuvVpSDQA5TBODvKQjlD9STcJOPc1fzQ
7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1200713/?fbclid=IwAR1w_WKTVVkm6B137KPoAj5MARw9141xQzV-7ExqkmT6QFqhMxTFPyZMgaI