Genetic Sequencing
- DNA sequencing is the process used to determine the precise sequence of nucleotides in a length of DNA.
- Sanger sequencing involves:
- The DNA to be sequenced is placed in 4 tubes and mixed with primers, DNA polymerase and free nucleotides
- In each of the tubes, chain-terminating nucleotides for one specific base were also added. The 4 tubes are then placed in a thermocycler and PCR begins
- DNA polymerase binds to the template DNA and DNA synthesis of the complementary strand began. Chain-terminating nucleotides were randomly incorporated into the growing DNA chains, stopping DNA synthesis
- The many cycles of PCR resulted in the production of thousands of fragments of DNA that all differed in length by one base, which are electrophoresed, and the sequence of DNA is determined.
- Pyrosequencing involves:
- The DNA is fragmented and separated into single-stranded DNA (ssDNA) and one of these strands is fixed to a flow cell (a plastic slide)
- The flow cell contains millions of DNA strands, each ssDNA acting as a template for the sequencing reaction, and they are each fixed in their own well in the flow cell
- The flow cell is then incubated in a reaction mixture containing primers, DNA polymerase, ATP sulfurylase, luciferase, apyrase, Adenosine 5’ phosphosulfate (APS) and luciferin
- The first reaction takes place by adding activated nucleotides that contain only one of the four bases A, G, C or T to the flow cell. If the first base of the template DNA is complementary to the base added, the nucleotide is then added into the DNA chain by DNA polymerase. This results in the release of diphosphate (PPi), also knows a pyrophosphate
- Pyrophosphate reacts with APS to form ATP, in a process catalysed by the ATP sulfurylase
- Luciferase then uses ATP to convert luciferin to oxyluciferin, generating visible light which is detected by a camera
- Any nucleotides that were not incorporated into the DNA are degraded by apyrase
- A different activated nucleotide is then washed across the flow cell resulting in another flash of light if incorporated
- This cycle repeats until all of the DNA fragments have been fully synthesised
Genetic Fingerprinting
- Genetic fingerprinting is a method used to produce a specific pattern of DNA bands from an individual’s genome.
- The non-coding regions of DNA contain short, repeating sequences called variable number tandem repeats (VNTRs).
- VNTRs are found at many locations in the genome. In every individual, they vary in length and the in the number of repeats at different loci. Therefore, the probability of two individuals having the same VNTRs is very low.
- The steps in DNA fingerprinting include:
- Extraction of DNA & amplification using PCR
- DNA digestion using specific restriction endonucleases, leaving the VNTRs intact
- Separation of DNA fragment by gel electrophoresis. Smaller fragments travel faster and therefore move further down the gel
- Hybridisation of the VNTRs at specific (complementary) base sequences with Radioactive or fluorescent DNA probes
- Development. The banding pattern can then be visualised as radiation, emitted by fragments, exposes X-ray film (placed over the gel) and reveals their final positions
- The DNA profiles can be compared to determine genetic relationships by looking for similarities in the banding pattern.
- DNA profiles can also be used in:
- Forensic science investigations- comparing the DNA profiles of suspects and DNA at the crime scene.
- Medical diagnosis- DNA profiles can identify individuals at risk of developing specific diseases, as some VNTRs are correlated with an increased risk of disease e.g. Huntington’s disease
- Animal and plant breeding- DNA profiles are used to prevent inbreeding by not breeding individuals with similar profiles
- Paternity determination- half the DNA profile of the child should match the father
Polymerase Chain Reaction (PCR)
- PCR is a method of amplifying DNA by artificial replication in vitro.
- It requires: DNA sample of around 10,000 base pairs, nucleotides, Taq polymerase (stable at high temperatures), primers complementary to 3’ of DNA sample and a thermocycler to carry out the automated process.
Electrophoresis
- Electrophoresis is the process of separating DNA fragments or other macromolecules, according to their size.
- The stages involved is as follows:
- DNA loading dye is added to the DNA samples, and it is pipetted into the wells in the agarose gel plate
- An electric current is applied across the plate, so DNA moves towards the anode. Smaller fragments travel faster
- Proteins can be separated according to their molecular mas if sodium dodecyl sulphate is added to give the proteins a uniform negative charge.
- The DNA bands can be visualised by using DNA probes, which are short sections of DNA that are complementary to a known DNA sequence. They can be fluorescent or radioactive.
- Microarrays can also be formed which contain a number of different probes on a fixed surface. This allows for the DNA to reveal the presence of mutated alleles if it matches the fixed probes.
Genetic Engineering
- Genetically modified organisms are organisms that have had their DNA altered through recombinant DNA technology.
- Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another.
- Transgenic organisms can successfully express a gene from any organism, as the genetic code and mechanism of protein production (transcription and translation) are universal.
- DNA fragments are created by:
- Using restriction endonucleases to cut at recognition sites near the desired gene
- Converting the mRNA of the desired gene to cDNA, using reverse transcriptase. Double stranded DNA is then synthesised using DNA polymerase
- Synthesising the gene using a gene machine. The gene sequence is determined by the primary protein structure
- The isolated gene is then modified by the addition of a promoter and a terminator region.
- A vector is used to transfer the isolated gene into a host cell. This is mainly a plasmid.
- Restriction endonucleases are used to cut plasmids open, creating sticky ends. The same endonuclease isolates the gene, so the sticky ends of the desired gene and the plasmid are complementary. DNA ligase joins them together
- To reintroduce the desired DNA into bacterial cells, the recombinant plasmid must pass through the cell surface membrane of a bacterial cell (transformation).
- Transformation involved mixing the bacteria and plasmids in a medium containing Ca^2+ ions, which increased membrane permeability. Changes in temperature also make the bacterial cell surface more permeable.
- The transformed host cells can be cultured as an in vivo method to amplify DNA fragments.
The Use of Genetically Modified Organisms (GMOs)
- The risk of GM bacteria can be reduced by modifying the bacteria so that they are unable to produce an essential nutrient or amino acid and cannot survive outside the lab
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Gene Therapy
- Gene therapy is the mechanism by which genetic diseases are treated or cured by masking the effect of a faulty allele through the insertion of a functional allele.
- Firstly, a healthy allele from healthy cell tissue is isolated. The allele is inserted into the cells using vectors.
- If the mutated allele is recessive, a dominant allele is inserted. If the mutated allele is dominant, DNA is inserted into the middle of the allele to silence it.
- Somatic therapy involves altering the alleles in body cells. The altered allele is not passed onto the offspring
- Germ-line therapy altering the alleles in the sex cells. The altered alleles are passed onto offspring
- Germ-line therapy has ethical concerns such as the potential of designer babies or the potential impact gene insertion could have on other genes.