DNA Profiling

What does every person have a unique combination of?

DNA in the chromosomes of their cells, unless they are an indentical twin or triplet.

What is the genome of an organism?

All the genetic information it contains - for eukaryotes, that is the DNA in the nucleus and the mitochondria combined.

What are chromosomes made up of?

Hundreds of millions of DNA base pairs, but genes only make up about 2% of the total DNA.

What are our genes known as?

Exons, coding DNA.

What happens to the mRNA following transcription?

The non-coding regions of DNA are removed before it is translated into a polypeptide chain known as introns.

What is satellite DNA?

Short sections of non-coding DNA (introns) that are repeated many times.

What is a minisatellite?

A region of non coding DNA composed of 20-50 base pairs that will be repeated 50-100 times.

What is a microsatellite?

A smaller region of just 2-4 bases repeated only 5-15 times.

Where do microsatellites appear, and how do they vary?

They always appear in the same positions on the chromosomes, but the number of repeats on each microsatellite varies between individuals, as different lengths of repeats are inherited from parents.

What is DNA profiling?

Producing an image of the patterns in the DNA of an individual.

What are the main stages in producing a DNA profile?

1. Extracting the DNA.

2. Cutting the DNA.

3. Separating DNA fragments.

4. Making DNA single-stranded.

5. Southern blotting.

6. Probing the DNA.

7. Visualising the profile.

What must the DNA be extracted from?

A tissue sample.

What is done to the DNA sample, so there is enough for analysis?

It is amplified, using PCR.

How is the DNA cut?

The DNA strands are cut into small fragments using restriction endonucleases. Each enzyme cuts DNA at a specific nucleotide sequence, knows as a restriction site. This produces fragments of different lengths, often from inron regions.

What is added after the DNA sample is cut?

Buffer solution, to maintain the pH.

How are DNA fragmented separated?

By gel electrophoresis. Charged DNA fragments move through gel in an electric current. Smaller fragments move further than larger ones.

How does the DNA become single stranded?

The electrophoresis gel is soaked in alkali, which separates double-stranded DNA into single strands.

What happens in southern blotting?

Single-stranded DNA fragments are transferred onto a membrane.

How is the DNA probed?

Either radioactive or fluorescent DNA probes are added, Probes are complimentary to specific DNA sequences. They bind to DNA by hybridisation, targeting microsatellite regions.

How do you visualise the DNA pofile?

In radioactive probes, the membrane is exposed to X-ray. In fluorescent probes, the membrane is exposed to fluorescent light. This produces a DNA profile.

What is PCR?

Polymerase chain reaction.

How does PCR work?

For the DNA sample to be amplified, an excess of the four nucleotide bases, small primer bases and DNA polymerase are mixed in a vial placed in a PCR machine.

What are the three stages in the PCR cycle?

• Denaturation.

• Annealing.

• Elongation.

What happens in Denaturation?

The temperature in the PCR machine is raised to around 95 degrees for 30s. This denatures the DNA, by breaking the hydrogen bonds between the bases, so the DNA strands separate.

What happens in annealing?

The temperature is decreased to around 55 degrees and the primers bind (anneal) to the ends of DNA. They are needed for the replication of the strands to occur.

What happens in elongation?

The temperature is increased to around 72 degrees for at least one minute, the optimum temperature for DNA polymerase. Taq polymerase is used to add bases to the primer, producing double stranded DNA that is identical to the original sequence.

What are the different uses of DNA profiling?

• Forensics - the profile is compared to that of a sample taken from a suspect.

• Maternity and paternity tests.

• Genetic diseases.

Where can DNA traces be obtained from?

Blood, semen, saliva, hair roots and skin cells.