Module 8: V9 - V12

Question 1

What are some major techniques of Molecular Biology?

Answer 1

‘cloning’ DNA into a vector - restriction enzymes, plasmids, transformation
amplifying DNA - PCR
sequencing DNA
there are also many techniques with RNA and protein

Question 2

What is required for the cloning of DNA?

Answer 2

cells and plasmids

Question 3

What is the role of a plasmid vector?

Answer 3

holds a piece of DNA inside the cell

Question 4

What sequences must a plasmid have?

Answer 4

pBR322 has an origin of replication (ori), resistance genes to ampicillin + tetracycline and restriction sites including EcoRI, PstI, BamHI and SalI

Question 5

What are restriction enzymes?

Answer 5

recognise short palindrome sequences and cut these sequences

Question 6

What type of ends do restriction enzymes create in the DNA?

Answer 6

some generate ‘sticky ends’ and ‘blunt ends’ for others

Question 7

What are compatible DNA ends able to do?

Answer 7

can be base-paired and then joined together by DNA ligase resulting in the formation of recombinant DNA

Question 8

Where do restriction enzymes originally come from?

Answer 8

bacteria which use these enzymes as viral defence proteins

Question 9

How are recombinant vectors made in the laboratory?

Answer 9

cloning vector is cleaved with restriction endonuclease (RE) -> DNA fragment of interest is obtained by cleaving chromosome with RE -> fragments are ligated to the prepared cloning vector -> recombinant vector

Question 10

How is recombinant DNA made from recombinant vectors?

Answer 10

recombinant vector is introduced into the host cell and propagation (cloning) of transformed cell produces many copies of recombinant DNA

Question 11

Why does propagation of transformed cells require growth in a medium which contains antibiotics?

Answer 11

to ensure only the transformed bacteria are producing DNA

Question 12

Which additional sequences are required in a plasmid to express a protein?

Answer 12

bacterial promoter (P) and operator (O) sequences, ribosome-binding site, polylinker, transcription-termination sequence and gene coding repressor that binds O and regulates P

Question 13

What are the possibilities once a protein is expressed?

Answer 13

express for functional assays, express in a model organism and express in human cells in cell culture

Question 14

What is the overall process of PCR?

Answer 14

1. heat briefly at 98˚C to denature DNA strands
2. cool to 58˚C to allow primers to form hydrogen bonds with ends of target sequence (annealing)
3. at 72˚C DNA polymerase adds nucleotides to the 3’ end of each primer (extension)
4. repeat

Question 15

Which enzyme is used in DNA polymerase and why?

Answer 15

Taq DNA polymerase because it originates from bacteria which grow in hydrothermal vents and therefore will not denature at 98˚C

Question 16

How many cycles of PCR are required to produce 1 billion copies of a target sequence?

Answer 16

30 cycles

Question 17

What can PCR be used for?

Answer 17

amplify / identify DNA from crime scene samples, amplify / identify presence of water-borne pathogens, to do DNA fingerprints for identity/paternity testing, to identify individuals in a population of animals

Question 18

What is RT-PCR?

Answer 18

when PCR is combined with another technique which allows for the identification of the level of gene expression

Question 19

What sort of sequences would you need on a piece of DNA so that a bacterial cell will hold it for you (and it would be maintained as the bacteria grew and divided)?

Answer 19

an origin of replication (ori), resistance genes to antibiotics and restriction sites

Question 20

What are all the things you know about PCR? Eg. what does it stand for, what is it used for? What are the reagents?

Answer 20

stands for polymerase chain reaction, used to amplify DNA, reagents include Taq polymerase, primers, free nucleotides and DNA strands

Question 21

What does it mean that nucleic acids bind with reverse complement? Can you draw this out and explain it? Can you think of any circumstances where nucleic acid binding is only reverse but not complement? Or any circumstances where nucleic acid binding is only complement but not reverse?

Answer 21

the reverse complement of a DNA sequence is formed by reversing the letters, interchanging A and T and interchanging C and G
+ strand is complementary but not reversed in regards to the - strand
mRNA is complementary and reversed in regards to the + strand