6.1.3: manipulating genomes Flashcards
what is dna sequencing?
process of determining the nucleic acid sequence
add about sangar sequencing
yeah bro dont forget :)
what is the method for dna profiling?
1) extract the dna
- from a mouth swab/saliva/blood/single hair/semen/bone sample etc
- dna needs to be purified because it is wrapped around histones, which need to be removed
2) digest the sample with enzymes
- cut with restriction enzymes
3) separation of the dna fragments: gel electrophoresis
- put into well in a block of gel, with some loading dye (make dna visible)
- alkaline buffer solution to help regulate ph and help carry electrical charge across gel
- electrical current = dna moves towards positive end
- smaller fragments move further (less resistance)
- sufficient time to let them fully separate out
- southern blotting = nylon membrane placed on top of cell, with absorbent paper on top
4) hybridisation
- dna probes added to look for mini/micro-satellites in dna
- probes = single-stranded dna used to detect presence of complementary nucleic acid sequences
5) radioactive probes viewed by x-ray and fluorescent viewed by uv
what is pcr?
- polymerase chain reaction
- “amplify” dna sample (making multiple copies for analysis)
what is needed for pcr?
- original, tiny dna sample
- excess of free nucleotides
- primers
- dna polymerase / tac polymerase
what are the steps for pcr?
1) denaturing phase (90-95 degrees for 30 seconds) - breaks the hydrogen bonds between the dna strands
2) annealing phase (55-60 degrees) - primer join to both ends of the 2 dna strands (have complementary ends)
3) extension phase (70-75 degrees for 1 minute) - tac polymerase adds bases to the primers, extending the complementary strands
= double stranded dna genetically identical to original sample
why might we not get as many copies of dna as expected using pcr?
- not enough primers
- primers may not attach to all dna strands
- insufficient nucleotides
- dna strands may re-join together, rather than with the primers
