Using gene sequencing and PCREdexcel A-Level Biology B: Revision notes

The genome is the complete set of genetic material (DNA) in a cell of an organism, including both genes and non-coding DNA. The human genome contains about 3 billion base pairs in a haploid set of chromosomes.

Knowing the genome sequence lets scientists locate genes, compare alleles between individuals and identify changes linked to disease. It is not the same as the proteome, which is the set of proteins an organism makes.

PCR amplifies a specific region of DNA to produce millions of copies from a very small sample, in a thermal cycler. The mixture contains the template DNA, primers, free nucleotides, Taq polymerase (a heat-stable DNA polymerase from a thermophilic bacterium) and buffer.

Each cycle has three steps:

1. Denaturation at about 95 °C: hydrogen bonds between the strands break, giving single strands.
2. Annealing at about 55 °C: primers bind to complementary sequences either side of the target.
3. Extension at about 72 °C: Taq polymerase adds nucleotides to the 3' end of each primer, making new complementary strands.

The number of copies doubles each cycle, so after $n$ cycles there are $2^n$ copies from a single molecule. After 30 cycles this is $2^{30} \approx 1.07 \times 10^{9}$ copies.

Once a gene has been amplified, its base sequence can be determined. Because the genetic code is known, the mRNA codons and so the amino acid sequence (primary structure) of the protein can be predicted.

Example: sickle cell disease. The healthy template triplet CTC gives the mRNA codon GAG (glutamic acid). A single base substitution to CAC gives GUG, which codes for valine. This changes the primary structure of haemoglobin and so the shape of the molecule.

Sequencing a person's gene and comparing it with the normal sequence can show whether they have, or carry, an allele linked to a genetically determined condition such as cystic fibrosis or sickle cell disease.

DNA profiling compares regions of the genome that vary between individuals. The usual targets are short tandem repeats (STRs): short sequences repeated a variable number of times. People differ in the number of repeats, so the fragments have different lengths.

1. DNA is extracted and the STR regions are amplified by PCR (essential for tiny samples such as a bloodstain).
2. The fragments are separated by gel electrophoresis: DNA is negatively charged, so it moves towards the positive electrode and smaller fragments move further.
3. The pattern of bands is the DNA profile.

A profile from the crime scene is compared with those of suspects. A match on all STRs makes a suspect very likely the source, as the chance of two unrelated people sharing a profile is very small but not zero.

A child inherits half of its DNA from each parent. In a paternity test, profiles are produced for the mother, child and the possible father.

• Bands in the child that match the mother's profile are taken to be maternal.
• Every remaining band in the child must have come from the father.
• A man is excluded if he lacks any of those paternal bands. If he has all of them he is very probably the father.

Example: child bands 1, 2, 3, 4 and mother bands 1, 2, 5. Bands 3 and 4 must be paternal, so a man with bands 3, 4 and 6 fits, but a man with bands 2, 5 and 7 is excluded.