Enzymes and restriction mapping Flashcards
What can we produce from genetic engineering?
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
What are nucleases?
enzymes that degrade nucleic acids by hydrolysing (breaking) phosphodiester bonds
What do ribonucleases do?
Degrade RNA
What do deoxiribonucleases do?
degrade DNA and are further subdivided into:
Exonucleases
-degrade DNA from end of the molecule
Endonucleases
-cleave DNA within nucleotide chain
What are restriction endonucleases expressed by?
Restriction endonucleases are expressed by bacteria (different types expressed from different bacteria)
What is the Benefit of restriction endonucleases in bacteria?
limit the transfer of nucleic acids from infecting phages (virus) into bacteria
Give the functions of restriction endonucleases.
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
Define palindromic.
the recognition sequence reads the same backwards and forwards
Which restriction nucleases produce overhangs?
- cleavage by EcoRI produces a 5’ overhang
- cleavage by KpnI produces a 3’ overhang
Which restriction nucleases produce blunt ends?
-cleavage by restriction enzyme AluI produces a blunt end
Which enzyme can reverse blunt-ends of DNA overhangs?
DNA polymerase
Restriction enzymes can be used to generate…
restriction maps
What is a restriction map?
a map of known restriction sites within a sequence of DNA
used as a reference to engineer plasmids
What is the Restriction site in sickle cell anaemia?
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
What is DNA ligase?
joins two DNA fragments together by forming a phosphodiester bond between them
What is the role of DNA polymersae?
synthesizes DNA in the 5’ to 3’ direction
What is required for DNA polymerase to carry out its functions?
DNA polymerase needs primers to facilitate the addition of nucleotides onto the daughter strand
What is DNA Polymerase used in the lab for?
- PCR Amplification
- Generation of probes
- Blunt-ending of DNA overhangs
What is the role of phosphatase?
hydrolyse (remove) a phosphate on DNA to prevent a phosphodiester bond between nucleotides
Where are phosphatase enzymes derived from?
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
What is needed for a cut plasmid DNA to be resealed?
needs phosphate groups on its ends to be resealed
What is the Use of phosphatase enzyme in the lab?
prevent cut plasmids from resealing by removing the phosphate group from each end to prevent resealing in a ligation reaction
What is the role of polynucleotide kinase?
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
What is the Use of polynucleotide kinase?
- 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
What are probes?
fragments of ssDNA (or RNA) 20-1000 bases in length, complimentary to the gene of interest
What is the Use of probes?
can be either radioactively or fluorescently labelled to be used in DNA hybridisation
What is the role of reverse transcriptase?
RNA dependent DNA polymerase which makes DNA copies from RNA molecules
Where is reverse transcriptase found?
retroviruses whose genomes are made of DNA
How does reverse transcriptase synthesize DNA?
synthesizes a DNA molecule complimentary to an mRNA template using dNTPs
What is needed for reverse transcriptase to bind to mRNA?
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
What are random primers?
- 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
What is an oligo-dT primer?
- will hybridise with adenine nucleotides in poly(A) tail of mRNA
- useful for cloning cDNA libraries, but some might not be full length
What is a Gene Specific Primer?
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
