DNA Fragment Amplification

Question 1

What is PCR?

Answer 1

An in vitro cloning method to produce many identical copies of a specific DNA sequence.

Question 2

What happens to the number of DNA molecules every PCR cycle?

Answer 2

Doubles

Question 3

Describe the process of PCR.

Answer 3

1. DNA sample is heated to around 95 degrees which causes hydrogen bonds to break and the double helix to unwind into two single strands
2. Temperature is then lowered to around 55 degrees which allows primers to attach (annealing) to the single stranded DNA
3. The temperature is then increased to around 72 degrees which allows heat stable DNA Polymerase to work
4. Free DNA nucleotides form complementary base pairs with bases on strands forming hydrogen bonds
5. DNA Polymerase joins adjacent nucleotides by phosphodiester bonds in condensation reactions

Question 4

What are some application of PCR?

Answer 4

• genetic fingerprinting
• detecting mutant genes
• confirming paternity
• establishing evolutionary relationships

Question 5

What is in vivo gene cloning?

Answer 5

Using bacteria to make copies of the gene

Question 6

What needs to be added to the isolated foreign gene depending on the process used for isolation?

Answer 6

1: mRNA to cDNA = promotor region
2: using restriction endonuclease = none
3: using gene machine = promotor and termination

Question 7

Describe ligation of isolated foreign genes.

Answer 7

• foreign gene is inserted into the vector (eg. Plasmids)
• plasmid is cut open using the same restriction enzyme that was used to isolate the foreign gene
• this creates sticky ends that are complementary to the sticky ends of the isolated foreign gene which join via hydrogen bonds
• ligase joins the nucleotides by phosphodiester bonds creating recombinant DNA

Question 8

How is transformed bacteria produced?

Answer 8

• host bacterial cells placed in ice cold calcium chloride solution
• plasmids (recombinant DNA) are added and the mixture is heated to around 40 degrees causing heat shock
• bacterial cells take in the recombinant DNA

Question 9

How are transformed bacteria identified?

Answer 9

Marker genes on the recombinant DNA

Question 10

How is transformed bacteria cultured?

Answer 10

• in industrial fermenters
• supplied with nutrients and oxygen
• binary fission so each new bacterial cells placed contains the foreign gene so can make the protein
• protein is extracted for use

Question 11

What are the advantages of in vitro and in vivo cloning?

Answer 11

In vitro:
- very sensitive
- quick
In vivo:
- cloned gene can be expressed as a protein
- reliable copying of genes
- low error rate

Question 12

What is the role of a primer?

Answer 12

• marks the starting point for TAQ Polymerase to begin working
• ensures only a specific section of the strand is amplified

Question 13

What is the advantage of using bacteria in in vivo cloning?

Answer 13

• replicate rapidly
• produce identical cells
• cheap