DNA profiling Flashcards
What is pcr
Polymerase chain reaction - a technique used to select a fragment of the dna you are interested in and produce millions of copies of it.
What is the process of PCR
1) a reaction mixture set up containing the DNA sample, free nucleotides, primers and DNA polymerase
2) mixture heated to 95°C to break the hydrogen bonds between the two strands of DNA. Luckily DNA polymerase to denature.
3) after separation!the mixture is cooled to 55°C so that the primers can bind
4) after binding, mixture heated to 72°C for DNA polymerase to work
5) DNA polymerase lines up free DNA nucleotides alongside each template strand and complimentary base pairing occurs
6) two new copies of the fragment DNA is formed and one cycle of PCR is complete.
7) cycle restarts (heated to 95°C) and now all 4 strands acts as templates and this continues
What is electrophoresis
A procedure that uses electrical current to separate DNA fragments, RNA fragments or proteins depending on their size.
What is electrophoresis performed with?
It is performed by Using agarose gel that has been poured into a gel tray and left to solidify. A row of wells is created at one end of the gel
How does electrophoresis occur with this gel tray?
1)The gel tray must be put into a gel box, making sure the end of the gel try with the wells is closest to the negative electrode of the gel box
2) then add a buffer solution to the reservoirs at the sides of the gel box so that the surface of the gel becomes covered in buffer solution
3) take fragmented dna samples using micro pipette and add same volume of loading dye to each sample as this makes them sink and easier to see
4) add a set volume of dna to the first well. And using a clean pipette add the same to the next well until all are full.
5) record which DNA sampl you have added to each well
6) put the lid on and connect gel box to power supply and set it to the required voltage.
7) DNA fragments are negatively charged so they will move towards the positive electrode at the far end of the gel. Small fragments move faster and further, so fragments separate according to size
8) let the gel run for 30 minutes then turn off power and remove the gel tray from the gel box and tip off any excess buffer solution
9) wearing gloves, stain the dna fragments by covering the surface if the growl with a staining solution then rinsing the gel with water. The bands of the different DNA fragments will now be visible. The size of dna fragments will now be visible.
What is the purpose of DNA sequencing
Allow us to quickly compare relations between species, and see if there’s links between genetic diseases and predict amino acid sequences… as well as classifying species
What is the Sanger termination technique
Extract DNA
Cut it into various lengths and amplify these fragments with PCR.
Pour fragments into 4 different solutions containing DNA nucleotides, DNA polymerase, Primers and a terminator base (either A,G,C,T)
Terminator bases can end the sequence at that base
Complimentary pairing could then be either from a normal base or a terminator base so we are going to have various lengths of DNA in each test tube ending in whatever terminator base was added
What is a faster way to sequence DNA
Massive parallel sequencing.
Can sequence many dna at the same time
What is placed in the 4 electrophoresis wells?
The 4 different solutions of containing either the A T G C terminators
How to view DNA after electrophoresis
Southern blotting - radioactive DNA probes and x rays
Green fluorescent protein and UV
What are uses of DNA profiling
Paternal Tests
Crime scene tests
Searching for genes that can trigger diseases
What are introns
Non coding DNA is most likely to be different from other people as exons are very similar in everyone, so would not produce unique profiles. They are not required for survival
