Enzymes And Restriction Mapping Flashcards
What are the uses of genetic engineering?
Transgenic organisms and recombinant proteins
Give some examples of transgenic organisms
Disease models and improved agricultural yields
Give some examples of recombinant proteins
Insulin, interferon and G-CSF
What does G-CSF do?
Promote formation of bone marrow in cancer patients
What do nucleases do?
Degrade nuclear acids by hydrolysing phosphodiesterase bonds
What degrades RNA?
Ribonuclease
What degrades DNA?
Deoxyribonuclease
What do restriction endonucleases do?
Limits the transfer of nucleic acids from infecting phages into bacteria
How do restriction endonucleases work?
Recognising a specific sequence and cutting it.
What is special about restriction endonuclease recognition sites?
4-8 bases in length and palindromic
What does DNA ligase do?
Joins DNA molecules from different sources to form recombinant DNA
What can SNPs do?
Create/ destroy restriction enzyme sites
What do you use the enzyme Dde1 to do?
Cleave when doing PCR for sickle cell anaemia testing
What is a restriction map?
A crude way of mapping an unknown molecule
What is a restriction map useful for?
Describing plasmids
How do you get a restriction map?
By performing gel electrophoresis
What are the main uses of DNA polymerases?
PCR amplification, generation of probes and blunt- ending of DNA
What are phosphatases used for?
Preventing cleaved plasmids from resealing
How do phosphatases work?
Hydrolysing a phosphate group off its substrate
How do polynucleotide kinases work?
Adds phosphate to the 5’ hydroxyl group of DNA or RNA
What are polynucleotide kinases used for?
To sensitively label DNA so that it can be traced using radioactively or fluorescently labelled ATP
What is a probe?
A fragment of single stranded DNA or RNA that is complementary to the gene of interest
How long are probes generally?
20-1000 bases
How does reverse transcriptase work?
RNA dependant DNA polymerase that is isolated from RNA containing retroviruses. Then synthesises a DNA molecule that is complementary to an mRNA template using dNTPs
What are the three types of primers for reverse transcription?
Random primers, oligoprimers and gene specific primers
What is good about using random primers for reverse transcription?
Complementary DNA up to 700 bases but will cover the length of all the RNA molecules
What is good about using oligoprimers for reverse transcription?
They’re useful for cloning cDNAs and their libraries but some might not be full length
