manipulating genomes

Question 1

what does a polymerase chain reaction (PCR) do

Answer 1

.it selects a fragment of DNA and amplifyies it to make millions of copies

Question 2

what are the stages for a PCR

Answer 2

.a mixture of the DNA sample, free nucleotides, primers and DNA polymerase is set up

.the mixture is heated to 95C to break the H bonds between the two strands of DNA

.the mixture is cooled to 50-65C so that the primers can bind to the strands

.the mixture is then heated to 72C so that the DNA polymerase can work at is optimum

.DNA polymerase lines up the DNA nucleotides next to each template strand and via complementary base pairing, new strands are formed

Question 3

what results from each cycle of the PCR

Answer 3

.each cycle forms double the amount of DNA

first one starts with 2 and ends with 4 strands

Question 4

what is electrophoresis

Answer 4

uses an electrical current to separate out DNA, RNA fragments or proteins

Question 5

process of electrophoresis

Answer 5

.put the gel tray into a gel box making sure that the end of the gel tray with the wells is closest to the negative electrode

.add buffer solution to the reservoirs at the sides of the box so that the surface of the gel becomes covered

.take the fragmented DNA and using a micropipette add the same volume of loading dye to each

.add a set vol of DNA sample to one of the wells

.repeat using the other DNA samples and use a clean micropipette each time

.record which DNA sample is in each well

.put a lid on the gel box and connect the leads to the power supply and turn on to required voltage

Question 6

what happens during electrophoresis

Answer 6

.a electrical current is passed through the gel
.DNA fragments are negatively charged and so move through the gel towards the anode
.smaller fragments will move faster and further = DNA fragments are separated by size

Question 7

what to do after electrophoresis

Answer 7

.turn off power supply
.remove gel tray and tip off an excess buffer solution
.wearing gloves, stain the DNA fragements by covering the surface of the gel with a staining agent
.rinse the gel with water

.the bands of the DNA fragments will now be visible

.size of DNA fragments are measured by bases e.g. ATCC = 4 bases
1000 bases = kilobase

Question 8

electrophoresis of protein

Answer 8

.same as DNA and RNA but the proteins are denatured by a chemical to ensure that they are all the same charge

Question 9

what is DNA sequences

Answer 9

determining the sequence of nucleotides within a DNA molecule

Question 10

what is the process for sequencing DNA

Answer 10

.DNA is mixed with primers, DNA polymerase, nucleotide bases and terminator bases

.the DNA is split into single strands and copied multiple times

.DNA polymerase add nucleotides to the template strand to start rebuilding new strands

.when the terminator base is added, synthesis stops with each terminator tagged with a fluorescent colour

.the DNA fragments are separated by size using either capillary sequencing or electrophoresis

.laser then detects the colours of the terminators to determine the sequence order

.computer software analyses the fragments to reconstruct the original DNA sequence

Question 11

what are the pros that comes from modern day technologies for DNA sequencing

Answer 11

.there are parallel sequencing

.there is increased speed

.there is reduced costs