1- Using gene sequencing

What is a genome?

The complete set of genetic information contained in the cells of an organism (all of an organism’s DNA, including mitochondrial/ chloroplast DNA).

What is DNA sequencing?

Identifying the base sequence of a DNA fragment.

What is a PCR and what does it do?

Polymerase chain reaction, it is used to amplify the DNA by making millions of copies of a given DNA sample.

Describe the reaction mixture in the first stage of a PCR.

Contains:

• The DNA fragment which will be amplified.

• Primers that are complementary to the start of the fragment.

• Free nucleotides which are complementary to the exposed bases.

• DNA polymerase to create the new DNA.

Describe the process of amplifying DNA fragments using a PCR.

1. A reaction mixture is set up by mixing the DNA sample, primers, free nucleotides and heat-stable DNA polymerase, which is the enzyme involved in creating new DNA stands.

2. The mixture is heated to 95 degrees C to break the H bonds between the complementary bases and to separate the two strands.

3. The mixture is then cooled to a temperature between 50-65 degrees, depending on the type of primers used, so the primers can bind to the stands (annealing).

4. Temperature is increased to about 70 degrees, as this is the temperature DNA polymerase works at. DNA polymerase creates a copy of the sample by complementary base pairing using the free nucleotides.

5. This cycle is repeated around 30 times and gives rise to an amount of DNA sufficient to create a DNA profile.

How can DNA sequencing be used in medicine?

To screen for heritable conditions. When the base sequence of a particular allele or gene is known, we can test a person’s DNA to see if that allele or gene is present.

How can DNA sequencing be used in forensics?

To compare DNA obtained during crime investigations against the DNA of victims or suspects, in order to identify them or discount them.

How (describe the method) is DNA profiling used to identify criminals and test paternity?

1. Fragments of DNA are cut with restriction endonuclease enzymes (either side of satellites).

2. These fragments are separated and visualised using gel electrophoresis- fragments are placed in wells in agarose gels and dyed with ethidium bromide so they fluoresce under UV light. A current is then applied to the gel. DNA is negative and fragments of different sizes move at different speeds according to mass so ‘bands’ appear.

3. Southern Blot- alkaline buffer solution added, nylon filter- dry absorbent material draws solution containing DNA fragments to the filter- fragments visible as ‘blots’. Gene probes (labelled complementary sequences that fluoresce or are radioactive) are added and bind with DNA (hybridisation).

4. ‘Blots’ compared and the number of satellites visualised as a graph (repeated sequences of DNA in introns are referred to as mini/ microsatellites depending on their size. The more closely related two people/ species are, the more similar the repeats are).

What other ways can DNA sequencing be used?

• To predict the amino acid sequence of proteins.

• To test relatedness of two individuals, including paternity testing.

How is DNA sequencing used to predict the amino acid sequence of proteins and determine possible links to genetically determined conditions?

• The DNA sample is divided into 4 separate sequencing reactions which contain all 4 standard nucleotides, DNA polymerase, primers required for replication and terminator nucleotides which have been fluorescently labelled for ease of identification.

• When a terminator nucleotide is incorporated into a growing chain, replication is terminated.

• DNA fragments of different lengths are produced across the reaction vessels.

• High-resolution gel electrophoresis is used to separate the fragments by size- single base differences can be seen.

• The fragments are visualised under UV light, enabling the base sequence to be read from the bottom of the gel upwards.