Enzymes and restriction mapping Flashcards
Recombinant proteins
Insulin
Interferon
G-CSF
Transgenic organisms
Disease models
Improved agricultural yields
Restriction, ligation & other enzymes
Nucleases
- Degrade nucleic acids by hydrolysing phosphodiester bonds
- Ribonuclease (RNase): degrade RNA
- Deoxyribonuclease (DNase): degrade DNA
Exonuclease
Degrade from end of molecule
Endonucleas
Cleave within nucleotide chain
Restriction endonucleases
Limit transfer of nucleic acids from infecting phages into bacteria.
What do restriction endonucleases do?
- recognise a specific sequence
- cut that sequence
Restriction enzymes catalyse the hydrolysis of phosphodiester bonds
Different restriction enzymes recognise …
Different specific DNA sequences
What are recognition sites?
4-8 base pairs in length, depending on the enzyme, and palindromic
Some nucleases
Produce an overhang or
Produce a blunt end
What are restriction enzymes needed for?
Cloning
Molecular diagnostics
Characterization of plasmids
What do restriction molecules allow?
DNA molecules from different souces can be joined
Restriction enzymes in molecular diagnostics
Sickle cell anaemia - Single nucleotide changes can create/destroy restriction enzyme sites
Ddel site
(5’CTNAG3’) is lost is sickle cell anaemia
Restriction maps
- Map of restriction sites within a molecule
- Crude way of mapping an unknown molecule
- Useful way of describing plasmids
DNA ligase
Repairs nicks in phosphodiester backbone
DNA polymerase
DNA synthesis in 5’ to 3’ direction
Why use DNA polymerase?
• PCR amplification
• Generation of probes.
• Blunt-ending of DNA overhangs.
The 2 DNA fragments can now be ligated.
Phosphatase
Hydrolyses a phosphate group off its substrate.
• Calf intestinal alkaline phosphatase
• Shrimp alkaline phosphatase
Why use a phosphatase?
To prevent cut plasmids from resealing.
Polynucleotide kinase
Phosphate from ATP to substrate
Polynucleotide kinase adds phosphate to 5’ hydoxyl group of DNA or RNA
Why use a polynucleotide kinase?
- 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
- fluorescently labeled ATP
Probes
- Fragment of ssDNA (or RNA)
- 20 – 1000 bases in length
- Complementary to the gene of interest
Reverse transcriptase
- RNA dependent DNA polymerase
- Isolated from RNA-containing retroviruses
- Synthesizes a DNA molecule complementary to a mRNA template using dNTPs
Priming for reverse trancription
- Random primers: cDNAs up to 700bp but will cover all of the length of all of the RNA molecules.
- Oligo(dT) primers: useful for cloning cDNAs and cDNA libraries, but some might not be full-length.
