manipulating genomes Flashcards
why are bioinformatics useful?
- format of information is universal
- facilitates access to large amounts of data
- genes can be put into clusters which show the same pattern of gene expression
what is a genome?
all the DNA of an organism including the genes that carry all the info for making a protein
what is a PCR? (polymerase chain reaction)
a biomedical technology that can be used to amplify a short length of DNA into thousands of millions of copies.
outline the stages of PCR
- Add DNA template to nucleotides, primer, and taq polymerase, and add to thermocycler.
- Mixture is then heated to 95C to separate two DNA strands.
- Mixture is cooled to 68C to allow primers to anneal.
- Mixture is heated to 72C to allow Taq Polymerase to add nucleotides to the new strand of DNA.
- Process is repeated until required amount of DNA is obtained.
why is the PCR heated to 95C?
expose the bases by separating hydrogen bonds.
why is the PCR heated to 68C
allows the primers to anneal and tells polymerase which parts need to copy.
why is the PCR heated to 72C?
optimum temperature for taq polymerase to function
Why is Taq polymerase used in the PCR?
- Adapted to live in hot environments.
- Enzymes do not denature at hot temperatures.
- Allows PCR to continue as a cycle without having to add more enzyme.
Why is PCR difficult to use?
- Too little DNA
- DNA damaged/fragmented
- DNA sample is contaminated
how is PCR different to natural DNA replication
- natural DNA works at 37C, PCR works at 95C
- DNA polymerase VS Taq polymerase
- PCR is smaller (has 10,000 base pairs)
how is PCR used in tissue typing
reduce risk of rejection of transplanted organs
how is PCR used in detection of encogenes
finding a particular mutation that has resulted in cancer to give tailored medicine
how is PCR used in forensics
amplifying small quantities found at crime scenes for genetic profiling
how is PCR used in identifying viral infections
amplifying small quantities of viral DNA in patient samples
Explain how PCR is used in genetic finger printing
PCR allows amplification of small quantities of DNA from fingerprinting which can be used for genetic profiling
which way will DNA move in electrophoresis and why?
it will move towards positive anode because DNA is always slightly negative
what is electrophoresis?
separation of DNA fragments depending on size, used to separate proteins by their size.
outline the steps of electrophoresis
- Restriction endonuclease cut DNA samples at specific recognition sites.
- Agarose gel is set up with alkaline buffer (provides resistance to movement)
- Dense loading dye is used to help add the digested DNA to the wells.
- Make sure not to pierce the bottom of the well with pipette!
- DNA samples are put in place and gel is left.
- DNA fragments move through the gel (smaller ones travel faster) towards the anode as DNA has an overall negative charge.
- Buffer is poured away, and dye is added to stain the fragments.
suggest 2 uses of electrophoresis
genetic finger printing
forensics
why do smaller fragments travel faster through the gel in electrophoresis?
lower molecule mass because lower number of base pairs, therefore less resistance to flow.
what is the function of the loading dye in electrophoresis?
help add digested DNA to the wells.
what is the problem of using electrophoresis?
not all proteins have a negative surface charge
what is the solution to the fact that not all proteins have a negative surface charge in electrophoresis?
heat protein to denature them and expose hydrophobic region. Use a charged detergent to equalise the surface charge to make sure all proteins are negatively charged. Separation will therefore be down to mass/length, and not charge.
what is DNA profiling?
the creation of a unique genetic finger printing of an individual
what are minisatellites (variable number tandem repeats VNTRs)
a sequence of 20-50 base pairs repeated hundreds of times.
how many base pairs are micro-satellites? what are they also referred to as?
2-4 base pairs - short tandem repeats (STRs)
state the (one word!!) procedure of DNA profiling
- extraction
- digestion
- separation
- hybridisation
- development
if restriction enzymes are cut at specific recognition sites, how does the number of VNTRs affect the size of DNA fragments produced after being cut at restrictions enzymes?
more VNTRs = larger fragments.
satellites always appear at the same position in the…
chromosome