Enzymes and restriction mapping

Question 1

What can we produce from genetic engineering?

Answer 1

Recombinant Proteins

• insulin
• interferon (antiviral)
• G-CSF (stimulates production of bone marrow in those undergoing radiotherapy)

Transgenic Organisms

• disease models (e.g. mouse models)
• improved agricultural yields

Question 2

What are nucleases?

Answer 2

enzymes that degrade nucleic acids by hydrolysing (breaking) phosphodiester bonds

Question 3

What do ribonucleases do?

Answer 3

Degrade RNA

Question 4

What do deoxiribonucleases do?

Answer 4

degrade DNA and are further subdivided into:

Exonucleases
-degrade DNA from end of the molecule

Endonucleases
-cleave DNA within nucleotide chain

Question 5

What are restriction endonucleases expressed by?

Answer 5

Restriction endonucleases are expressed by bacteria (different types expressed from different bacteria)

Question 6

What is the Benefit of restriction endonucleases in bacteria?

Answer 6

limit the transfer of nucleic acids from infecting phages (virus) into bacteria

Question 7

Give the functions of restriction endonucleases.

Answer 7

Two functions:

1) recognise specific DNA sequence
2) cut that sequence by catalysing the hydrolysis of phosphodiester bonds

*different restriction enzymes recognise different specific DNA sequences

Question 8

Define palindromic.

Answer 8

the recognition sequence reads the same backwards and forwards

Question 9

Which restriction nucleases produce overhangs?

Answer 9

• cleavage by EcoRI produces a 5’ overhang

- cleavage by KpnI produces a 3’ overhang

Question 10

Which restriction nucleases produce blunt ends?

Answer 10

-cleavage by restriction enzyme AluI produces a blunt end

Question 11

Which enzyme can reverse blunt-ends of DNA overhangs?

Answer 11

DNA polymerase

Question 12

Restriction enzymes can be used to generate…

Answer 12

restriction maps

Question 13

What is a restriction map?

Answer 13

a map of known restriction sites within a sequence of DNA

used as a reference to engineer plasmids

Question 14

What is the Restriction site in sickle cell anaemia?

Answer 14

In SCA, the restriction site (5’CTGAG3’) for enzyme DdeI enzyme is changed due to a single nucleotide change (A to T), and therefore enzyme can’t recognise sequence and there is no restriction site

Question 15

What is DNA ligase?

Answer 15

joins two DNA fragments together by forming a phosphodiester bond between them

Question 16

What is the role of DNA polymersae?

Answer 16

synthesizes DNA in the 5’ to 3’ direction

Question 17

What is required for DNA polymerase to carry out its functions?

Answer 17

DNA polymerase needs primers to facilitate the addition of nucleotides onto the daughter strand

Question 18

What is DNA Polymerase used in the lab for?

Answer 18

• PCR Amplification
• Generation of probes
• Blunt-ending of DNA overhangs

Question 19

What is the role of phosphatase?

Answer 19

hydrolyse (remove) a phosphate on DNA to prevent a phosphodiester bond between nucleotides

Question 20

Where are phosphatase enzymes derived from?

Answer 20

historically, from calf intestines (calf intestinal alkaline phosphatase)

nowadays, it is purified from prawns (shrimp alkaline phosphatase) as it is a lot better to work with in the lab

Question 21

What is needed for a cut plasmid DNA to be resealed?

Answer 21

needs phosphate groups on its ends to be resealed

Question 22

What is the Use of phosphatase enzyme in the lab?

Answer 22

prevent cut plasmids from resealing by removing the phosphate group from each end to prevent resealing in a ligation reaction

Question 23

What is the role of polynucleotide kinase?

Answer 23

does the opposite of a phosphatase and adds a phosphate group from aTP onto a substrate
-adds a phosphate to 5’OH group of DNA or RNA

Question 24

What is the Use of polynucleotide kinase?

Answer 24

• to phosphorylate chemically synthesized DNA so that it can be ligated to another fragment
• to radioactively or fluorescently label DNA with ATP so that it can be traced

Question 25

What are probes?

Answer 25

fragments of ssDNA (or RNA) 20-1000 bases in length, complimentary to the gene of interest

Question 26

What is the Use of probes?

Answer 26

can be either radioactively or fluorescently labelled to be used in DNA hybridisation

Question 27

What is the role of reverse transcriptase?

Answer 27

RNA dependent DNA polymerase which makes DNA copies from RNA molecules

Question 28

Where is reverse transcriptase found?

Answer 28

retroviruses whose genomes are made of DNA

Question 29

How does reverse transcriptase synthesize DNA?

Answer 29

synthesizes a DNA molecule complimentary to an mRNA template using dNTPs

Question 30

What is needed for reverse transcriptase to bind to mRNA?

Answer 30

primer needs to bind to mRNA molecule so that reverse transcriptase can bind

there are different ways to prime the reverse transcriptase enzyme to work:

• random primer
• oligo(dT) primer
• gene specific primer

Question 31

What are random primers?

Answer 31

• exonucleotides which consist of 6 nucleotides
• will randomly bind at any position on the mRNA molecule
• cDNAs up to 700bp (short fragments) but will cover all of the length of the RNA molecule since the primers bind throughout the mRNA molecule

Question 32

What is an oligo-dT primer?

Answer 32

• will hybridise with adenine nucleotides in poly(A) tail of mRNA
• useful for cloning cDNA libraries, but some might not be full length

Question 33

What is a Gene Specific Primer?

Answer 33

when cloning a gene and the enzyme can’t reach the end of an RNA molecule because it is way too long, a gene specific primer is designed for the gene you want to clone

-RNA codes for the stop codon