W2 - Enzyme And Restriction Mapping

Question 1

What are some examples of Recombinant proteins?

Answer 1

• Insulin - diabetes
• Interferon - antiviral
• G-CSF - given to radiotherapy patients to promote Haemopoietic stem cells.

Question 2

What are transgenic organisms?

Answer 2

• Mice for example can be disease models
• Plants for eg can be used to improve agricultural yields

Question 3

What are nucleases?

Answer 3

Degrades nucleic acid by hydrolysing phosphodiester bonds.

2 types:
1) Ribonuclease(RNase) -> degrades RNA
2) Deoxyribonuclease (DNase) -> degrade DNA

Question 4

What is an exonuclease?

Answer 4

Degrades from end of DNA molecule

Question 5

What are endonucleases?

Answer 5

Cleaves within nucleotide chain of DNA molecule. 

Question 6

What are restriction endonucleases?

Answer 6

Limits transfer of nucleic acid from infecting phages into bacteria.
Different enzymes come from different species of bacteria.

Enzymes that recognize a specific DNA sequence, called a restriction site, and cleave the DNA within or adjacent to that site.

Question 7

What do restriction endonucleases do?

Answer 7

1) recognise a specific sequence
2) Cut that sequence

Recognition sites of 4-8 base pairs in length, depending on the enzyme, and palindromic.

Question 8

What are overhangs?

Answer 8

Enzymes like EcoRI produces 5’ overhangs and eg. KpnI produces 3’ overhangs.
These are a stretch of unpaired nucleotides in the end of a DNA molecule - mostly palindromic.

Question 9

What are blunt ends?

Answer 9

Eg, Alu I - recognises a certain base pair sequence and cleave it right in the middle creating blunt ends…

Question 10

What are restriction enzymes used in?

Answer 10

• Cloning
• Molecular Diagnostics
• Characterisation of plasmids

Question 11

How do DNA molecules from different sources join together in cloning?

Answer 11

Eg. A human DNA can be cleaved and placed in a plasmid or fuse it with bacterial DNA (which is also restricted via enzymes). The overhangs in these DNA will be complementary through hybridisation and will join each other.

DNA ligase will make a Phosphodiester bond between these two different DNA molecules.

The result is a recombinant DNA molecule.

Question 12

How are restriction enzymes used in diagnostics?

Answer 12

Eg. Sickle cell anaemia - restriction enzymes are used for PCR. Ddel is used as an enzyme which recognises the CTGAG site in a normal chain. Ddel site is lost in sickle cell anaemia.

The point of having 2 Ddel sites is that they want to make sure the enzyme works in labs.

Question 13

What are restriction maps?

Answer 13

It is a map of restriction sides within a molecule. A crude way of mapping an unknown molecule. Useful way of describing plasmids - can test if it’s the correct plasmids before conducting an experiment !
Gel electrophoresis…

You can compare it to the size standards for fragments to create the map.

Double digestion.

Question 14

Why do we use DNA polymerase and PCR?

Answer 14

PCR amplification, generation of probes and Blunt ending of DNA overhangs. 

Question 15

Why do they remove phosphatase in recombinant DNA?

Answer 15

They remove the phosphate group of a substrate. This prevents cut plasmids from resealing.

Question 16

What does polynucleotide Kinase do?

Answer 16

This is the opposite of phosphatase. This adds a phosphate group taken from ATP.

ATP + Substrate — kinase—> ADP+ phosphorylated substrate

polynucleotide kinase adds phosphate to 5’ hydroxyl group of DNA or RNA.

Question 17

Why would we use A polynucleotide Kinase?

Answer 17

To phosphorylate chemically synthesised DNA so that it can be ligated to another fragment.

To sensitively label DNA so that it can be traced using:
- radioactively labelled ATP
- Fluorescently labelled ATP

Question 18

What is Reverse Transcriptase?

Answer 18

RNA dependent DNA polymerase. Isolated from RNA containing retroviruses. Synthesised a DNA molecule complimentary to an mRNA template using dNTPs. 

Uses an RNA template and makes DNA copies.

Question 19

What is priming for reverse transcription?

Answer 19

Random Primers: cDNA up to 700bp but will cover all of the length of all RNA molecules.
Oligo primers: Useful for cloning cDNA and cDNA libraries - some may not be full length.

To initiate reverse transcription, reverse transcriptases require a short DNA oligonucleotide called a primer to bind to its complementary sequences on the RNA template and serve as a starting point for synthesis of a new strand.