Lecture 5 - PCR And DNA Manipulation

Question 1

What does PCR stand for? And who came up with it?

Answer 1

polymer chain reaction and Mullis 1993

Question 2

What is required for PCR?

Answer 2

Template DNA, primers, DNTP, a buffer and Taq polymerase (from thermos aquatious bacteria that survive at high temp)

Question 3

What is the process of PCR?

Answer 3

1. Identification of the region to be amplified.
2. Denaturation: heat to 94° to break the hydrogen bonds to create a template strand.
3. Annealing: primers added at 50 to 65° and anneal to DNA, polymerise attaches and starts copying the DNA
4. Extension: at 72° Taq polymerase is a optimum temp and the fragment is extended.

Question 4

How does agarose gel electrophoresis work?

Answer 4

Current is applied to gel and the DNA moves toward the positive electrode due to its negative phosphate backbone
The separation is dependent on the shape of DNA as well as its size smaller fragments move faster and further than large ones

Question 5

What are the limitations of PCR?

Answer 5

Sequence information is required to design primers, there’s a limit on the length of the amplified fragment. There’s a high error rate. It is very sensitive to reaction conditions, and tiny amounts of contaminating DNA will also be amplified

Question 6

What is required for Sanger sequencing?

Answer 6

DNA polymerase, DNTPs ,template DNA, and a primer

Question 7

What is the process of Sanger sequencing?

Answer 7

1. Small amounts of didexoyribonucleic triphosphates and lots of dNTPs are added
2. Products such as ACTAC ACTACG ACTACGT are formed
3. Products are loaded onto a capillary gel and separated depending on size
4. Sequence is formed

Question 8

What might cut DNA?

Answer 8

Restriction endonucleases that cut at palindromic, specific places, creating sticky or straight ends

Question 9

What does DNA ligase do?

Answer 9

Forms phosphodiester bonds, requiring ATP

Question 10

What is it called when an organism possesses a gene from another?

Answer 10

Transgenic