Using Gene Sequencing

Question 1

What is the purpose of PCR?

Answer 1

To amplify a section of DNA (make more copies) to carry out gene sequencing

Question 2

How do you carry out PCR?

Answer 2

You mix the sample with everything it needs to replicate and put it in a PCR machine
Heated to 95c for the hydrogen bonds to break
Cooled to 55c so primers can bind
Heated to 72c which is the optimum tempreture for DNA polymerase
Steps repeated around 30 times

Question 3

What is put in the PCR machine with the sample?

Answer 3

DNA polymerase from a bacteria
Primers
Free nucleotides

Question 4

What kind of DNA polymerase is used in PCR and why?

Answer 4

Enzymes from a bacteria that lives in hot springs as it won’t denature when the sample is heated

Question 5

Why must the DNA sample be heated to 95c in PCR?

Answer 5

So the hydrogen bonds holding the strands of DNA together break

Question 6

Why must the DNA sample be colled to 55c in PCR?

Answer 6

So that primers are able to bind to the DNA

Question 7

Why must the DNA sample be heated to 72c in PCR?

Answer 7

This is the tempreture that the DNA polymerase works best at and helps free nucleotides to bind to the single strands of DNA

Question 8

What are introns?

Answer 8

Large, non-coding sections of DNA that are removed before mRNA is translated into proteins

Question 9

What are exons?

Answer 9

Coding sections of DNA

Question 10

What is DNA sequencing?

Answer 10

Where you find the order of the bases in the sequence of DNA

Question 11

What is the process of DNA sequencing?

Answer 11

DNA strands are chopped into smaller peices and go through PCR
Labelled terminator bases are added where synthesis stopps after that base
We can use the length of the chains to determine what order they come in, the shortest coming first

Question 12

What are satellites?

Answer 12

DNA sequences in the introns that are repeating. The number of repeats is unique to each person

Question 13

How is a DNA profile produced?

Answer 13

The satellites are cut from DNA using restriction endonucleases and then seperated with gel electrophoresis
Southern Blotting is then carried out

Question 14

How is gel electrophoresis used to make a DNA profile?

Answer 14

DNA put in the gel and then a current is passed through it. DNA fragments move towards the positive anode at different rates depending on size

Question 15

During electrophoresis, does DNA move towards the anode or the cathode?

Answer 15

Anode

Question 16

How is southern blotting carried out to produce a DNA profile?

Answer 16

An alkeline solution is placed in the gel and a nylon filter put on top. This draws up the gel and leaves the DNA fragments in place
Complimentry gene probes are then added to highlight the target genes