Session 15 ILOs - Molecular Techniques Flashcards
Explain the process of Southern blotting
Southern blotting is the process of DNA hybridisation and gel electrophoresis
Gel electrophoresis:
- Involves putting the DNA in a gel matrix and the DNA will move towards the anode, but the largest DNA fragments will move the slowest towards the anode and the smallest fragments will move the fastest due to least resistance.
- This separates each of the DNA fragments
DNA hybridisation:
- Denature the DNA by heating to 90 degrees which separates DNA into 2 single strands of DNA
- Then add a complimentary primer which binds to the complimentary sequence
- Cool the DNA which allows the DNA to reanneal and renature
Southern blotting (Gel electrophoresis THEN DNA hybridisation):
- DNA is transferred from the agarose gel to the nitrocellulose sheet
- Fluoresent probe added (or radioactive probe) and then you use fluorescence imaging to reveal the DNA fragments
Outline applications of other hybridisation-based technologies
Other hybridisation-based technologies:
Fluorescent in situ hybridisation
PCR
Explain the molecular basis of PCR
Polymerase chain reaction:
- DNA mixture heated up to around 90 degrees to denature the DNA
- Cooled slightly to 50 degrees to allow the primers to anneal to the single strand sequence
- Heat again back to 75 degrees so DNA polymerase becomes active and we make copies of DNA again
- This process is a cycle and is what allows amplification of the target DNA sequence = exponential increase in DNA!
Outline the use of RT-PCR and the appropriate controls that should be used
RT-PCR is used to determine whether a gene is expressed by detecting gene expression (rather than DNA sequencing).
- It involes reverse transcription of RNA into DNA (cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR)
Appropriate control for RT-PCR:
- Positive control could be a known sample of DNA, shows that the primers have attached to the DNA strand
- Negative control, a sample without DNA, shows if contamination of the PCR experiment with foreign DNA has occurred
Outline the mechanism of Sanger sequencing
Sanger sequencing:
- Based on the use of deoxyribose nucleic acid analog, which have a hydrogen group on the 3’ position, rather than a OH group
- This means that it is still recognised by DNA polymerase, however, it is unable to form a hydrogen bond in the sugar phosphate backbone, therefore it will stop chain elongation
- By introducing these analogs, there is still a 50% chance that a normal nucleotide is incorporated, so there is a lot of variation in the DNA sequences produced
- Can separate the fragments out by capillary electrophoresis
Explain the principles behind agarose gel electrophoresis
Agarose gel electrophoresis is a way of separating DNA fragments, using the charged nature of DNA
- Gel electrophoresis involves a gel matrix, a buffer solution, a power supply and a method of staining or detection
- DNA fragments are placed in loading wells near the negative electrode and when the power supply is turned on, this causes the DNA fragments to move towards the anion due to their innate negative charge
- Smaller DNA fragments move fastest through the matrix due to their size and larger fragments move more slowly
- This separates out the DNA fragments by size
- DNA can be visualised by adding a ethidium bromide
Outline the basic principles of SDS-PAGE
SDS-page:
- SDS-page involves a gel matrix, a buffer solution, a power supply and a method of staining or detection
- SDS breaks down the non-covalent interactions within the protein to remove the secondary and tertiary structures (single polypeptide chains)
- Proteins are placed in loading wells near the negative electrode and when the power supply is turned on, this causes the protein chains to move towards the anion due to their uniform negative charge (due to coating of SDS)
- The protein polypeptide chains are separated on SIZE only (native PAGE is different) - smaller proteins move fastest through the matrix due to their size and larger proteins move more slowly
Outline the mechanism of FISH
Fluorescent in situ hybridisation (FISH):
- FISH involves the process of hybridisation, to denature the DNA in order to bind a fluorescent probe in situ, into a cell
Outline how region- and chromosome-specific
probes can be used
Region specific:
- FISH can identify mutations in individual chromosomes using region specific probs
Chromosome-specific:
- FISH can also can identify aneuploidy with chromosome-specific probes (e.g. can identify trisomy 21), but it can also identify translocations between chromosomes
Outline how chromosome painting can be used
Chromosome painting is a form of chromosome specific FISH technique, where a chromosome-specific probe is used to identify different chromosomes
This can be used for Karyotyping e.g. for studying tumours
