8. Enzyme and Restriction Mapping

Question 1

Examples of recombinant proteins and effect transgenic organisms produced from genetic engineering.

Answer 1

• Recombinant proteins
- Insulin
- Interferon ~ produced by cells that have been effected by viruses. These induce an anti-viral defence response in cells.
- G-CSF ~ Granulocyte-colony stimulating factor. Given to patients that are undergoing radiotherapy. This stimulates the production of the cells that have died from the bone marrow and thus the cells that have died from the blood system, including haemological defence cells.
• Transgenic organisms
- Disease models ~ e.g. a mouse that mimic a particular human’s disease
- Improved agricultural yields

Question 2

What are nucleases? and what are the different types?

Answer 2

•Degrade nucleic acids by hydrolysing phosphodiester bonds
•There are 2 types:
1) Ribonuclease (RNase): degrade RNA

2) Deoxyribonuclease (DNase): degrade DNA. Can either be the following:
- Exonuclease: degrade from end of molecule
- Endonuclease: cleave within nucleotide chain

Question 3

What do restriction endonucleases do? and why are they called restriction?

Answer 3

• They do 2 things:

 1) Restriction enzymes recognise specific DNA sequences
 2) Restriction enzymes catalyse the hydrolysis of phosphodiester bonds

• They are called restriction as they prevent a bacteria from being infected by viruses (phages)
• There are many different enzymes from different bacteria.

Question 4

What are features of the recognition sites? and how often do they occur?

Answer 4

• They are 4-8 base pairs in length, (depending on the enzyme)
• They are palindromic
• A 4 base recognition sequence occurs every 4x4x4x4 = 256 bases
• A 6 base recognition sequence occurs every 4x4x4x4x4x4 = 4096 bases

Question 5

What types of ends do nucleases produce?

Answer 5

• Leave an overhang

* Or a blunt end

Question 6

What are restriction maps?

Answer 6

• They are maps of restriction sites within a molecule
• Crude way of mapping an unknown molecule
• Useful way of describing plasmids

Question 7

How do you separate DNA fragments? and how is the size of the fragments determined?

Answer 7

• Separated by charge in the gel
• Fragments are placed in the well and a current is applied and the fragments will move towards the positive charge
• Smaller fragments run faster and lighter fragments run slower
• Compare the fragments with the DNA ladder
• More than one fragment = more than one enzyme cut the DNA fragment

Question 8

How did the DNA sequence change to cause SCA? How did this change the restriction site?

Answer 8

Point substitution at position 6. Adenine was swapped with T, causing the AA glutamine to be swapped with valine. Caused a defective beta globin gene.

•Single nucleotide changes can create/destroy restriction enzyme sites - in this cases, the DdeI (name of the enzyme) site (5’CTNAG3’) is lost in SCA, and so this site cannot be cleaved.

Question 9

What is a recombinant DNA molecule?

Answer 9

When DNA molecules from different sources are joined together by DNA ligase.
•DNA ligase makes coupling phosphodiester bonds between 2 different DNA fragments.

Question 10

Why use a DNA polymerase when making a recombinant DNA fragment?

Answer 10

• PCR amplification
• Generation of probes.
• Overhangs may not be complementary so DNA pol will fill in the end of the overhang and form a blunt end, and not the 2 DNA fragments can be ligated.

Question 11

What is phosphatase and where does it come from? and why should it be used for producing recombinant DNA?

Answer 11

• The enzyme remove the phosphate group from the 5’ end.
• Initially, this enzyme used to come from calf intestinal alkaline phosphatase - but now from shrimp alkaline phosphatase as it is easier and cheaper
• It prevents the resealing of cut plasmids - as DNA ligase needs the phosphate to form the phosphodiester bond.

Question 12

What does a polynucleotide kinase do? and why should this be used for making a recombinant DNA?

Answer 12

•Kinase: phosphate from ATP to substrate
ATP + Substrate -> ADP + phosphorylated-substrate
• Polynucleotide kinase adds phosphate to 5’ hydroxyl group of DNA or RNA

•To phosphorylate chemically synthesized DNA so that it can be ligated to another fragment.
•To sensitively label DNA so that it can be traced
using:
- radioactively labeled ATP added
- fluorescently labeled ATP added

Question 13

Where are reverse transcriptase found? and what does it require to work?

Answer 13

• They are not found in eukaryotes - only expressed in retroviruses with their genome made of RNA
• It requires a primer on the mRNA template. - It is then able to add dNTPs and form a cDNA molecule.

Question 14

What are the different types of priming for reverse transcription?

Answer 14

• Random primer - cDNAs up to 700bp but will cover all of the length of all of the RNA molecules.
• Oligo (dT) primer - useful for cloning cDNAs and cDNA libraries, but some might not be full-length.
• Gene specific primer - design a primer that is gene specific
• (I think relates to oligo primer) If you want to code a gene, the way to do it is to purify the RNAs in the cells and you know that genes that code for a protein are polyadenylated and you would prime the polyA tail and reverse transcriptase will work. - reverse transcriptase will copy the full length of your fragment (but there is a physical limit – e.g. for a very large gene)