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Dna Fingerprinting Steps Involved In Critical Thinking

DNA Fingerprinting Definition

DNA fingerprinting – sometimes called “DNA testing” or “DNA profiling” – is a method used to identify living things based on samples of their DNA.

Early DNA fingerprinting was developed in the years before the whole human genome had been sequenced. Instead of looking at the whole sequence of a person’s DNA, these techniques look at the presence or absence of common markers that can be quickly and easily identified.

The best markers for use in quick and easy DNA profiling are those which can be reliably identified using common restriction enzymes, but which vary greatly between individuals.

For this purpose, scientists use repeat sequences – portions of DNA that have the same sequence so they can be identified by the same restriction enzymes, but which repeat a different number of times in different people.

Types of repeats used in DNA profiling include Variable Number Tandem Repeats (VNTRs), especially short tandem repeats (STRs), which are also referred to by scientists as “microsatellites” or “minisatellites.”

The term “satellite” may seem confusing – it comes from the fact that, when DNA is centrifuged, these “satellite” repeating regions float above the rest of the DNA.

By looking to see how many copies of each VNTR a person possesses, scientists can “fingerprint” them with a high degree of accuracy.

A DNA fingerprint looks something like the columns on the paper below. On this paper, each dark band represents a fragment of VNTRs – and each column is a different tissue sample. A match would be indicated by two columns whose VNTRs patterns matched precisely.

DNA fingerprinting is different from genetic testing, in which a DNA sample is tested to see if it contains genes for inherited diseases or other traits.

Function of DNA Fingerprinting

DNA fingerprinting is frequently used in criminal investigations to determine whether blood or tissue samples found at crime scenes could belong to a given suspect.

This technology is also used in paternity tests, where comparison of DNA markers can show whether a child could have inherited their markers from the suspected father.

In science, DNA fingerprinting is used in the story of plant and animal populations to determine how closely related species and populations are, and to track their spread over time.

This ability to look directly at an organism’s gene markers has revolutionized our understanding of zoology, botany, agriculture, and even human history.

DNA Fingerprinting Steps

Extracting DNA from Cells

To perform DNA fingerprinting, you must first have a DNA sample!

In order to procure this, a sample containing genetic material must be treated with different chemicals. Common sample types used today include blood and cheek swabs.

These samples must be treated with a series of chemicals to break open cell membranes, expose the DNA sample, and remove unwanted components – such as lipids and proteins – until relatively pure DNA emerges.

PCR Amplification (Optional)

If the amount of DNA in a sample is small, scientists may wish to perform PCR – Polymerase Chain Reaction – amplification of the sample.

PCR is an ingenuous technology which essentially mimics the process of DNA replication carried out by cells. Nucleotides and DNA polymerase enzymes are added, along with “primer” pieces of DNA which will bind to the sample DNA and give the polymerases a starting point.

PCR “cycles” can be repeated until the sample DNA has been copied many times in the lab if necessary.

Treatment with Restriction Enzymes

Once sufficient DNA has been isolated, and amplified if necessary, it must be cut with restriction enzymes to isolate the VNTRs.

Restriction enzymes are enzymes that attach to specific DNA sequences and create breaks in the DNA strands.

Bacterial cells use restriction enzymes for protection against DNA viruses which may invade the cell and hijack its machinery, but scientists have taken advantage of these special enzymes and used them to make DNA profiling and even genetic engineering possible.

In genetic engineering, DNA is cut up with restriction enzymes and then “sewn” back together by ligases to create new, recombinant DNA sequences.

In DNA profiling, however, only the cutting part is needed. Once the DNA has been cut to isolate the VNTRs, it’s time to run the resulting DNA fragments on a gel to see how long they are!

Gel Electrophoresis

Gel electrophoresis is a brilliant technology that separates molecules by size. The “gel” in question is a material that molecules can pass through, but only at a slow speed.

Just as air resistance slows a big truck more than it does a motorcycle, the resistance offered by the electrophoresis gel slows large molecules down more than small ones. The effect of the gel is so precise that scientists can tell exactly how big a molecule is by seeing how far it moves within a given gel in a set amount of time.

In this case, measuring the size of the DNA fragments from the sample that has been treated with restriction enzyme will tell scientists how many copies of each VTNR repeat the sample DNA contains.

It’s called “electrophoresis” because, to make the molecules move through the gel, an electrical current is applied. Because the sugar-phosphate backbone of the DNA has a negative electrical charge, the electrical current tugs the DNA along with it through the gel.

By looking at how many DNA fragments the restriction enzymes produced and the sizes of these fragments, the scientists can “fingerprint” the DNA donor.

Transfer onto Southern Blot

Now that the DNA fragments have been separated by size, they must be transferred to a medium where scientists can “read” and record the results of the electrophoresis.

To do this, scientists treat the gel with a weak acid, which breaks up the DNA fragments into individual nucleic acids that will more easily rub off onto paper.

They then “blot” the DNA fragments onto nitrocellulose paper, which fixes them in place.

Treatment with Radioactive Probe

Now that the DNA is fixed onto the blot paper, it is treated with a special probe chemical that sticks to the desired DNA fragments. This chemical is radioactive, which means that it will create a visible record when exposed to X-ray paper.

This method of blotting DNA fragments onto nitrocellulose paper and then treating it with a radioactive probe was discovered by a scientist name Ed Southern – hence the name “Southern blot.”

Amusingly, the fact that the Southern blot is named after a scientist has nothing to do with directions didn’t stop scientists from naming similar methods “northern” and “western” blots in honor of the Southern blot.

X-Ray Film Exposure

The last step of the process is to turn the information from the DNA fragments into a visible record. This is done by exposing the blot paper, with its radioactive DNA bands, to X-ray film.

X-ray film is “developed” by radiation, just like camera film is developed by visible light, resulting in a visual record of the pattern produced by the person’s DNA “fingerprint.”

To ensure a clear imprint, scientists often leave the X-ray film exposed to the weakly radioactive Southern blot paper for a day or more.

Once the image has been developed and fixed to prevent further light exposure from changing the image, this “fingerprint” can be used to determine if two DNA samples are the same or similar!

Quiz

1. Which of the following is NOT a use of DNA profiling?
A. Determining if two DNA samples come from the same person.
B. Determining if a child could have inherited their genes from a suspected father.
C. Determining whether a person has a given genetic disease.
D. None of the above.

Answer to Question #1

C is correct. DNA profiling looks at Variable Number Tandem Repeats (VNTRs), which are non-coding regions of DNA. VNTRs do not contain information about what genetic diseases or other traits a person has.

2. You have a DNA sample that you need to profile, but the amount of DNA is so small that you’re concerned the results might not be clear. What do you do?
A. Use extra radioactive chemicals on the Southern blot.
B. Use PCR to create copies of the DNA sample before running the electrophoresis.
C. Expose the X-ray for a week instead of a day at the end of the process.
D. All of the above.

Answer to Question #2

B is correct. The best way to ensure clear and reliable results with a small DNA sample is to amplify it using PCR.

3. Which of the following would result in an inaccurate or unusable DNA profile if it happened during the profiling procedure?
A. Skin cells from an investigating policeman get into the DNA sample from a crime scene before the sample is run.
B. The restriction enzymes had expired and did not work on the sample.
C. The power went out before the electrophoresis procedure was finished.
D. All of the above.

Answer to Question #3

D is correct. All of the above occurrences could result in incorrect or unusable DNA profile results.

Reference

  • A. (2015, March 31). A beginner’s guide to DNA fingerprinting. Retrieved May 11, 2017, from http://www2.le.ac.uk/offices/press/for-journalists/code-of-a-killer-1/a-beginners-guide-to-dna-fingerprinting
  • DNA migration in gel electrophoresis. (n.d.). Retrieved May 11, 2017, from http://scienceprimer.com/dna-migration-gel-electrophoresis

Dr Ed Hollox from the Department of Genetics and Science Advisor to Code of a Killer explains DNA fingerprinting

Seven 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
  • Yes - we've got the result!

= ECSTASY!

Extraction of DNA

All cells (except red blood cells) in all living creatures contain 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.

Yes - we've got the result!

The development process is similar to a traditional photograph.

A hybridisation oven

The glass tubes are placed horizontally in the oven on a wheel which moves slowly around. In the early days of DNA fingerprinting, instead of a hybridisation oven, Tupperware containers were used for 65 degree Celsius stage, and the paper was washed in plastic seed trays.

The film cassette is taken into the dark room and opened. The film can be either held with a gloved hand or placed in a metal frame. It is then dunked in three chambers “developer”, “stop” and “fix”, in the same way as traditional photograph developing. The bands appear slowly in the developer – you take it out occasionally and check it by holding it up against the red light – if the intensity of the bands is good then you dunk in “stop” then “fix”.

The developed film is then taken to the lab and examined on a white light box (horizontally placed on a lab bench, not vertically, not like “House”). The name and date, and details of the samples would be written in pen.

The key to the DNA fingerprint is the probe, the radioactive bit of DNA that identifies lots of fragments that contain the “minisatellite repeats”. These repeats have the 33 letters of DNA that are used in the probe but repeated lots of times. The number of repeats differ between different people. So the DNA fragment sizes with these different sized-repeats are different for different people – hence different black bands on a film for different people.

Some notes on PGM testing

We are familiar with blood groups A B and O to distinguish people. It’s best to think of PGM as another blood group with three types: PGM1, PGM2 and PGM2-1. It has the advantage over blood groups in that it can by typed on semen as well as blood – I suspect this was why it was chosen for the screen because the PGM type of the murderer was known from the semen.

The science behind PGM is interesting. PGM stands for phosphoglucomutase, and is an enzyme with different variants that is on the surface of red blood cells and sperm (and other cells). It is a protein, with two variants 1 and 2 so that an individual can be just 1, just 2, or both 1 and 2. There are also subtypes plus and minus, so that each individual can be 1+, 1-, 1+1-, 2+, 2-, 2+2-, 2+1+, 2+1-, or 2-1+. The forensic team would have used a gel separation technique called isoelectric focusing and polyacrylamide, not agarose gels, to separate the different PGM variants. This was cutting-edge forensic science at the time, but based on proteins not DNA. Like DNA fingerprinting, PGM testing is now not used in forensic investigations, being replaced by more modern DNA profiling techniques, but still rely on the same scientific principles.

Source: ITV Press Centre

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