Molecular Biology Week 3

Question 1

Name three types of enzymes that are used in a molecular biology lab?

Answer 1

Polymerase, Nucleases, Ligase

Question 2

What do Polymerases do?

Answer 2

Polymerases create polymers of nucleic acids ex: RNA and DNA

Question 3

How do polymerases

make new DNA or RNA?

Answer 3

New DNA/RNA is made using an existing DNA or RNA as a template of info for bp rules.

Question 4

Give an example for each DNA and RNA for a polymerase in vivo.

Answer 4

DNA ; rep and rep

RNA: transcription

Question 5

What are nucleases capable of doing?

Answer 5

They are capable of cleaving phosphodiester bonds between nucleotide (phos, sug and b). They can be both endonucleases and exonucleases (5’-3’ or 3’ to 5’)

Question 6

What is an example of a nucleases in vivo?

Answer 6

DNA rep and apoptosis

Question 7

What do DNA ligases do?

Answer 7

Ligases join DNA strands together by being a catalyst in the formation of a phosphodiester bond.

Question 8

Give an example of a ligases in vitro.

Answer 8

Cloning

Question 9

What is the biological function of restriction endonucleases in the bacteria that produce them?

Answer 9

Is to recognise one or more restriction sites and to make a double stranded cut in the DNA molecule. This cut occurs at the restriction site and occurs in an orderly pattern.

Question 10

What are the two different types of cuts made by RE and distingush between the two of them.

Answer 10

Pvull, allul are examples of blunt/protruding ends this involves the cleaving of both strands at SAME point of DNA. Uniform edges with NO overhangs.
Cutting of two strands of DNA at differnt points are called staggered cuts, this results in the generaton of blunt ends these are also known as staggered or sticky ends with complimentary overhangs.

Question 11

What does a restriction site look like and what is it called with the mirrored bp?

Answer 11

5’ - G - A - A- T - T - C - 3’ Palindromes

Question 12

What is the importance of the single stranded overhang created in some RE particularly useful?

Answer 12

Produces highly reactive sticky ends on the brokwn ends which are helpful for genetic engineering and cloning as these ends can stick together for long enough for the enzyme ligase to bind the two strands together.

Question 13

How are RE named?

Answer 13

Strain, Haemophilius Influenzae = HIND III
First three letters - abbreviate genus(H) and species (IN)
Fourth letter - comes from the bacterial strain designation (D)
Roman numerals indicates the order in which restriction enzymes were discovered in a particular strain (I, II, III)

Question 14

What is the difference between an RE and an exonuclease?

Answer 14

Exonucleases remove nucleotides from the ends of a nucleic acids molecule 5’ or 3’ end.
An exonuclease never produces internal cuts in the DNA

Question 15

What are the two forms of DNA replication?

Answer 15

Mitosis, Binary fission (bacteria)

Question 16

Since DNA double helix strands are complementary what does this mean in terms of the process of DNA replication?

Answer 16

These strands can be the template for DNA replication, the process is semi-conservative asa each daughter duplex has one parental strand and one new strand

Question 17

What are the two other theories proposed about DNA replication other than that of being semi-conservative.

Answer 17

Can either be fully conservative or dispersive

Question 18

Whos experiments proved that the process was definitely semi-conservative?

Answer 18

Meselson-Stahl

Question 19

Describe the three postulated models of DNA replication.

Answer 19

Conservative, both old,
Semi-conservative, new and old.
Dispersive, old + new parts added.

Question 20

Describe the Meselson Stahl Experiment.

Answer 20

They grew E.coli in heavy nitrogen 15 , gre also in nitrogen 14. Mixed together. First gen (time 0) all heavy 100%, next gen (20 min) 100% hybrid), after that the samples were half hybrid half light. No heavy DNA. Showed that DNA replication uses templates, splits, reads, adds new complementary strand.

Question 21

What direction does DNA polymerase catalyse in and starts at which part?

Answer 21

5’ to 3’ direction, DNA polymerase can only add a nucleotide to a free 3’ OH.

Question 22

Is DNA polymerase template dependent?

Answer 22

Yes it is, it reads the sequence of bases on the template strands and inserts the appropriate complementary base into the new strand.

Question 23

What are the sites in the DNA knows as where the replication begins?

Answer 23

Origins of Replication

Question 24

What enzyme must proceed first to initiate the process of replication? What shape does this produce in the DNA

Answer 24

Helicase enzymes must unwind the double helix structure of the DNA. gradually This produces a replication fork.

Question 25

What did Okazaki deduce?

Answer 25

That while one strand is being synthesised, the fork opening this is called the leading strand, the other is made discontinuously as short segments of DNA in the 5’ to 3’ direction are joined together to make a new strand called the lagging strand. The short fragments being joined onto this strand are called Okazaki fragments.

Question 26

DNA replication is ——– discontinuous.

Answer 26

self-discontinuous.

Question 27

What is the primer enzyme that is used in the initiation of DNA synthesis?

Answer 27

Primase enzyme to the 3’ OH which nucleotides are added to, these are the okazaki fragments that are then subsequently are plugged in

Question 28

What removes the primase enzyme and fills in the gaps?

Answer 28

Exonucleases remove the primers, DNA ligase then joins the 2 strands together. DNA polymerase fills the gaps.