dna profiling

Question 1

techniques for studying genes

Answer 1

PCR chain reaction
Cutting out DNA fragments using restriction enzymes
Gel electropheris

Question 2

explain PCR

Answer 2

used to select a fragment of DNA9of bit needed) and amplify it to make copies Inn just a few hours
Step 1: A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase. Primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want. DNA polymerase is an enzyme that creates new DNA strands.
Step2: The DNA mixture is heated to 95 °C to break the hydrogen bonds between the two strands of DNA. DNA polymerase doesn’t denature even at this high temperature — this is important as it means many cyc be carried out without having to use new enzymes each time.the mixture is then cooled to 50-65• so primers can bind/anneal to the strands.
Step3:reaction mixture if heated to 72• so DNA polymerase can work.DNA polymerase lines up free DNA nucleotides alongside each template strand. Complimentary base pairing means new complementary strands are formed .
Step 4: Two new copies of the fragment of DNA are formed and one cycle of PCR is complete. Then the cycle starts again — the mixture is heated to 95 °C and this time all four strands (two original and two new) are used as templates.

As shown below each PCR cycle doubles the amount of DNA eg 1st cycle =2x2=4 dna fragments,2nd cycle= 4 x 2=8 dna fragments

Answer 3

As well as PCR, another way to get a DNA fragment from an organism’s DNA is by using restriction enzymes. Here’s how they work:
Some sections of DNA have palindromic sequences of nucleotides.
These sequences consist of antiparallel base pairs (base pairs that read the same in opposite directions)

Restriction enzymes recognise specific palindromic sequences
(known as recognition sequences) and cut (digest) the DNA at these places.
- Different restriction enzymes cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to an enzymes active site
Eg restriction enzymes ECORI cuts at GAATTC
Eg restriction enzyme Hind11 cuts at AAGCTT

If recognition sequences are present at either side of the DNA
fragment you want, you can use restriction enzymes to separate it from the rest of the DNA — see Figure 3. The DNA sample is incubated with the specific restriction enzyme, which cuts the DNA fragment via a hydrolysis reaction.
Sometimes the cut leaves sticky ends — small tails of unpaired bases at each end of the fragment. Sticky ends can be used to bind (anneal) the DNA
sequences (there’s more about this on pages 212-213).
fragment to another piece of DNA that has sticky ends with complementary sequences

Question 5

Electrophoresis

Answer 5

Electrophoresis is a procedure that uses an electrical current to seperate out DNA fragments,RNA fragments or proteins depending on their size

Electrophoresis is commonly performed 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. To perform electrophoresis, firstly you need to put the gel tray into a gel box (or tank). You need to make sure the end of the gel tray with the wells is closest to the negative electrode on the gel box. Then add buffer solution to the reservoirs at the sides of the gel box so that the surface of the gel becomes covered in the buffer solution

Stage 2: Take your fragmented DNA samples and, using a micropipette, add the same volume of loading dye to each — loading dye helps the samples to sink to the bottom of the wells and makes them easier to see.
Next add a set volume (e.g. 10 ul) of a DNA sample to the first well.
You have to be really careful when adding the samples to the wells - make sure the tip of your micropipette is in the buffer solution and just above the opening of the well.Don’t stick the tip of the micropipette too far into the well or you could pierce the bottom of it. Then repeat this process and add the same volume of each of your other DNA samples to other wells in the gel. Use a clean micropipette tip each time. Make sure you record which DNA sample you have added to each well.
Stage 3

Put the lid on the gel box and connect the leads from the gel box to the power supply. Then turn on the power supply and set it to the required voltage, e.g.
100 V. This causes an electrical current to be passed through the gel.
DNA fragments are negatively charged, so they’ll move through the gel towards the positive electrode at the far end of the gel (called the anode).
Small DNA fragments move faster and travel further through the gel, so the DNA fragments will separate according to size.
Let the gel run for about 30 minutes (or until the dye is about 2 cm from the end of the gel), then turn off the power supply. Remove the gel tray from the gel box and tip off any excess buffer solution. Wearing gloves, stain the DNA fragments by covering the surface of the gel with a staining solution then rinsing the gel with water. The bands of the different DNA fragments will now be visible