- Created by: lizzie
- Created on: 12-12-12 22:00
The Importance of DNA
When a murder has been carried out it is both important to find out who carried out the murder and who has been murdered, to be able to inform their spouse as well as get some background information on the victim (as it may help the case).
You should know that DNA influences protein synthesis. The DNA is copied during transcription by the RNA nucleotides (found in the cytoplasm) and leaves the nucleus as a strand of mRNA. It arrives at the ribosome where the start codon signals for tRNA to start picking up amino acids that code for the codon. The tRNA translates the mRNA by binding to the complimentary bases. Meanwhile the amino acids they are carrying are bound together by enzymes to form a peptide bond.
In order to translate these amino acids, 3 bases on the RNA known as codons must transcribe to the corresponding tRNA. There are exceptions to this with the stop and start codons, which don’t carry any amino acids just instructions for the tRNA to start or stop coming.
Codons in DNA DO NOT overlap when they are being translated.
As economical as this would be (as 4 bases could code for 2 amino acids resulting in shorter mRNA strands), it would be impractical and potentially harmful. When DNA is being copied, occasionally the body makes mistakes, and a base of an amino acid may be changed. If DNA overlapped this mutation would affect 2 amino acids instead of just one resulting in a change in the active site of the amino acid changing its function completely – which could have serious complications.
It also means that Leucine (UUA/UUG) would only ever produced after phenylalanine because leucine only has codons starting with UU. This would restrict the possible combinations of proteins we could make as well as their function due to the specificity of the active site.
When we are looking at the bases it's very clear to see that the first and second codons limit what the amino acid could actually be. This may make the last base seem rather redundant, but if each amino acid was made by 1 codon, if there was a mistake in copying it would change the entire protein structure and function.
If there is a mutation with the current system to the last codon base, then there’s a chance it may not have any effect, as it may still code for the same amino acid.
For example if serine (AGU) changed to (AGC) it would still make the same protein structure.
Transcription Of DNA
DNA is unzipped with enzymes such as Helicase and DNA polymerase.
The free floating nucleotides in the nucleus attach to only 1 of the DNA strands. This strand is called the sense strand. The other is called the antisense.
After complimentary RNA mononucleotides attach to the DNA and RNA polymerase binds the mononucleotides together to form one strand of mRNA. REMEMBER - RNA is complimentary to DNA except that it has Uracil.
The mRNA is small enough to pass through the nuclear pore and carry the genetic information to the ribosome to begin translation.
Translation Of DNA
Another type of nucleotide tRNA is used in translation.
Each tRNA molecule has a unit of 3 bases called the anti codon because they are complimentary to the mRNA codons. One the opposite side of the tRNA is the corresponding amino acid. Each amino acid has its own tRNA molecule because of the correspondence to the anti-codon.
The mRNA strand lines up at the ribsomes with the start codon being read first. The tRNA anticodon binds to the mRNA codons via hydrogen bonds.
Neighboring amino acids form peptide bonds with the help of enzymes. This continues until the stop codon.
Preparation Of DNA
In order for a DNA profile to be made DNA is taken from any source of biological tissue of an animal or plant. Usually if dealing with a crime investigation it would come from a cheek swab, or white blood cells in a blood smear or sperm after sexual assault.
The sample of DNA is broken down in a buffer solution made of salt and detergent as this disrupts the cell membrane leaving the particles suspended in the cytoplasm.
To separate the DNA from all the other particles we put it in a centrifuge.
Protease enzymes are also added to remove proteins.
After all this, cold ethanol is added to precipitate out the DNA. This isn’t the end as the DNA will undergo further washing in buffer solution.
DNA and Identification
The human genome contains 20 000 to 25 000 genes and only 2% of these code for proteins and genes - these are called exons.
Of the rest of the DNA over 90% is made up of interons (sometimes referred to as short tandem repeats) and other repetitive coding regions all between the active gene coding. Their function is not fully understood but like genes they are inherited.
Back in the day, they used to be called junk DNA because there wasn’t any use for them before DNA profiling. Because these interons are inherited from parents they are very specific and unlike genes such as those used to code for proteins can only be found between families not the entire species.
What are Introns
Introns are short sequences of DNA that are repeated many times to for micro and mini satellites.
The difference between the 2 is the length of the bases and the information that can be deduced from them.
Mini satellites are bigger than microsatellites are bigger than micro satellites as they have a sequence of 20 to 50 bases that is repeated 50 to 100 times.
(ATGCGGG.....) n > 50
A micro satellite has only 2 – 4 bases that are repeated 5 to 15 times.
(AGC ) n < 15
Microsatellites are analysed during paternity cases and are an indicator of similarities between populations, i.e. race, regions. Whereas mini-satellites are used, (in conjunction with micro satellites), to construct a genetic fingerprint of individuals.
The prepared DNA is chopped up with fragments using special enzymes known as restriction enzymes (or more correctly restriction endonuclease).
The enzymes are found in bacteria where their function is to cut up invading Viral DNA.
There are many different restriction enzymes. Each type cutting the DNA molecule at different specific base sequences known as recognition sites.
Restriction enzymes are really amazing because they only cut DNA at the satellites. This leaves us the short tandem repeats of the DNA strand.
The DNA is separated using gel electrophoresis which is basically a variation of chromatography.
The DNA fragments are placed in wells of agrose or polyacrylamide gel. This acts like a medium for the DNA fragments to travel across.
As well as acting like a medium for the DNA the gel contains a dye that binds to the DNA fragments in the gel. This dye will fluoresce the DNA bands under UV light.
In the wells there is also a buffering solution that maintains the pH of the gel electrophoresis and an indicator dye that moves slightly faster than the DNA so that the current can be turned off before the sample runs off the end.
An electric current is passed through the electrophoresis. The DNA fragments move toward the positive anode because of the negative charge on the phosphate group in the DNA. The fragments move at different rates according to their size.
Once electrophoresis is done the done the DNA is placed under UV light and identified.
Southern blotting is the transfer of the DNA fragment pattern to a nylon or nitrocellulose paper. This is done so that the DNA can continue to be used and also to compare our sample DNA with other DNA samples. When it’s on the gel this isn’t possible because the gel dries out and the DNA fragments evaporate, before transfer a DNA probe is added to the well so that our target DNA shows up clearly when transferred.
To transfer the DNA an alkaline buffer solution is added to the gel after electrophoresis and the nylon filter placed over it. An absorbent piece of paper is placed over it to draw the DNA to the nylon filter. The alkaline solution denatures the DNA fragments so the strands separate and the base sequence are exposed. The DNA probes bind to these exposed bases.
Using Gene Probes
Gene probes are short DNA sequences that are complimentary to specific sequences that we are looking for.
Each probe is labelled with either a radioactive element or a florescent marker
When the DNA is being transferred from the gel to the nylon filter the gene probes are added so that they bind with complimentary DNA strands.
This is helpful because it arks the DNA making it show up under UV light or blacken under X-ray
This process is called hybridisation as the DNA strands have come from different sources
The sample DNA is immobilised by the filter – this allows the mobile DNA probe to bind to the complimentary parts of the DNA. The DNA probe only binds to specific STR’s so identification is fairly easy with this method.
Since we are analysing different DNA samples if the probe picks up the same STR from both samples it tells us that there is a match.
Polymerase Chain Reaction (Preparation)
DNA profiling needs 1μg of DNA which is equivalent to DNA from about 10 000 human cells. In a crime investigation there may only be a small sample to work with.
This is where the polymerase chain reaction comes in. The main purpose of it is to replicate as much DNA as is needed for the DNA profiling to be done.
In a test tube we put:
a sample of DNA which we are amplifying, DNA polymerase (which will bind the complimentary strand later on), primers (small sequences that attach themselves just before the STR’s we want to amplify and act like a signal to the DNA polymerase telling them to start adding complimentary nucleotides past a certain point) and of course the four nucleotides which the DNA polymerase will bind to the DNA.
Polymerase Chain Reaction
The mixture is heated to 90 – 95 C to separate the hydrogen bonds between the DNA sample making 2 DNA strands (usually referred to as 5’ 3’ strand)
The mixture is then cooled to 50 – 60 C so that the primers bind (or if we’re being scientific ANNEAL) to the single DNA strands.
The mixture is then heated to 75 C which is the optimum temperature for the dna polymerase enzymes to build complimentary strands for the DNA.
All of these steps are repeated 30 times to give 1 billion copies of the original DNA, and it usually take 3 hours. All of which are just heating and cooling.