Enzymes and Restriction Mapping Flashcards
Types of genetic engineering
Recominant proteins
Transgenic organisms
Examples of recombinant proteins
Insulin
Interferon
G-CSF
What is interferon?
Produced by cells that have been infected by viruses, induces anti-viral response in cells, given to patients with Hep C infection.
What is G-CSF?
Granulocyte-colony stimulating factor given to patients that are undergoing radiotherapy
Examples of use of transgenic organisms
Disease models
Improved agricultural yields
Nucleases
Degrade nucleic acids by hydrolysis (cleaving) phosphodiester bonds
Ribonuclease
RNAse - degrade RNA
Deoxyribonuclease
DNAse - degrade DNA
Exonuclease
degrade from end of molecule
Endonuclease
Cleave with nucleotide chain
Why is restriction important?
Limit transfer of nucleic acids from infecting phages (viruses) into bacteria.
What are restriction enzymes used for?
They were initially used to prevent the infection of bacteria by viruses (phages) infecting bacteria. Removing the viral DNA is done via a restriction enzyme that would cleave it into different pieces.
How was the use of restriction enzymes first identify?
First identified in E.coli and is called Restriction enzyme 1. EcoR1 binds and recognises a specific DNA sequence GAATTC.
It cleaves to form a 5’ overhang on both sides. This cleavage also leaves a phosphate group at the 5’ end of the molecule. On the 3’ end, a OH- group is left. This is specific to EcoR1.
What are restriction sites?
A 4-8 base pairs in length, depending on the enzyme, and palindromic.
Define palindromic
Can be read in both directions
How often does a 4 base recognition sequence occur?
4 to the power of 4 = 256 bases
How often does a 6 base recognition sequence occur?
4 to the power of 6 = 4096 bases
What do some nucleases produce when they cut sequences?
An overhang - EcoR1 produces 5’ and Kpn1 produces 3’ overhangs.
Some nucleases produce a blunt end - Alu1 recognises the DNA sequence and cleaves to produce blunt ends. It recognises 4 base pairs and digests the DNA.
What are restriction maps?
Map of restriction sites within a molecule where restriction enzyme sites are.
Why is restriction mapping used?
It is a crude way of mapping an unknown molecule when sharing data it can be used in other labs.
It is also useful way of describing plasmids and what they look like.
How many restriction enzyme sites does linear DNA have?
Only have one restriction enzyme site.
How many restriction enzyme sites does plasmid DNA have?
2 or more restriction enzyme sites.
What is restriction enzymes done in molecular diagnostics?
Can be used in sickle cell anaemia - there is a mutation at codon 6 which causes a change in the amino acid.
What can single nucleotide changes do? Give examples
It can create/destroy restriction enzyme sites. For example, in sickle cell anaemia, DdeI site (5’CTNAG3’) is lost to SCA. This means that a person with SCA has no DdeI site.
