Session 12: Introduction to Molecular Techniques Flashcards
Name three techniques that facilitate the analysis of human DNA
Restriction endonucleases
Cloning of DNA
DNA probes
What are restriction endonucleases?
Enzymes that are able to cut huge ds DNA molecules into defined fragments
What does cloning of DNA enable us to do?
Amplify specific nucleotide sequences
What do DNA probes enable us to do?
Identify and manipulate the nucleotide sequence of interest
Why do bacteria produce endonucleases?
To protect their own DNA by recognising and degrading foreign DNA
Restriction enzymes cleave dsDNA so as to produce __________ on one end and a _________ on the other
3’ OH group
5’ Phosphate group
TaqI is a restriction enzyme that forms staggered cuts that produce what?
“Sticky” ends
What are “sticky” ends that are produced by restriction endonucleases?
The resulting fragments of single stranded DNA produced after cleavage are complementary to each other
HaeIII produces fragments that have “blunt” ends, what does this mean?
The fragments produce ends that are double-stranded and therefore do not form hydrogen bonds with each other
What does DNA ligase do?
Covalently joins “sticky” ends of DNA fragments together
What is DNA gel electrophoresis?
A technique that allows us to separate DNA fragments based on their size or shape
How does DNA gel electrophoresis work?
Samples are loaded into a buffered gel and more towards the positively charged electrode due to their negative charge
The smaller fragments travel further than the larger fragments
DNA is ________ charged and will move towards the _________ if placed in an electric field
Negatively
Anode (+)
What 4 things are required for gel electrophoresis?
1) Gel
2) Buffer
3) Power supply
4) Stain/detection
What is the purpose of the buffer used in DNA gel electrophoresis?
To keep the pH constant and allow charge on the DNA samples across the gel
Give four reasons why we may use restriction analysis
To investigate the size of DNA fragments
To investigate mutations
To investigate DNA variation e.g. DNA fingerprinting
To clone DNA
What are the four basic steps of gene cloning?
Isolate the relevant gene of interest
Insert gene of interest into a plasmid vector
Introduce recombinant DNA molecule into suitable host cells
Identify and isolate the clone containing DNA of interest
Why might DNA cloning be important?
To clone useful proteins such as insulin
To find out what genes do
Genetic screening
Gene therapy (Potentially?)
When cloning eukaryotic genes would we overcome the fact that eukaryotic DNA contains both introns and exons?
Clone using the mRNA and reverse transcribe it to produce cDNA before placing into plasmid vector
What is Polymerase Chain Reaction (PCR)?
Process by which target DNA is amplified exponentially using forward and reverse primers to define the region to be copied
What might PCR be used for?
To amplify specific regions of a DNA fragment
To investigate single base pair mutations
To investigate small deletions or insertions
To investigate variation, genetic relationships
What are the three cyclical steps of PCR and their temperatures?
1) Denature at 95oC
2) Anneal at 55oC
3) Polymerise at 75oC
What are the types of DNA hybridisation?
Southern blotting (DNA) Northern blotting (RNA)
Why would we use Southern Hybridisation?
Looking at:
- gene structure (deletions, duplications) (CF)
- gene expansions, triplet repeats (Huntington’s)
- mutations in genetic tests (Sickle cell)
- Variation (Genetic fingerprinting)
Do the DNA probes used in blotting have to have 100% similarity to the target sequence?
No, they do not have to have 100% complimentarity to the sequence
Do probes have to completely align with the target sequence?
No, they don’t have to align, they can overlap and still detect the target sequence
Do probes affect the position of the target sequence as it appears as banding on the gel?
No, no affect on position of banding we detect on the gel
When we want to separate a protein based on their size, why do we first need to denature the protein in SDS?
To break the hydrogen bonds so that we have the protein as a linear polypeptide chain and they have a similar charge to mass ratio so are only separated based on their size and not its charge or shape
When are 2D blots particularly useful?
When analysing the whole cell or whole tissue
What are the stages in involved in Southern blotting?
- DNA gel electrophoresis
- Transfer of fragments to a membrane
- Hybridisation of a probe to detect specific piece of DNA
- Visualisation of labelled probe
Explain how Sanger Chain Termination works
dideoxyNTPs can be used along with labelled primers, initiate DNA synthesis and analyse the encorporation of the ddNTPs in the DNA to work out the nucleotide sequence of a DNA strand
Enzyme assays can be used to do what?
Determine the rate of an enzyme-dependent reaction
What does ELISA measure?
The concentration of proteins in a solution
