Enzymes and Restriction Mapping Flashcards
What are transgenic organisms?
Disease models e.g. transgenic mice
Improved agricultural yields through GM plants
What are nucleases? What are the two types?
Enzymes that degrade nucleic acids by hydrolysing phosphodiester bonds
Two types of nucleases-
Ribonuclease (RNase): degrade RNA
Deoxyribonuclease (DNase): degrade DNA
- Exonuclease: degrade from end of molecule
- Endonuclease: cleave within nucleotide chain
What are restriction endonucleases and what do they do?
Restriction: limit transfer of nucleic acids from infecting phages into bacteria.
Many different enzymes from different bacteria
Do two things:
1. recognise a specific sequence
2. cut that sequence
How do restriction enzymes work?
- Restriction enzymes recognise specific DNA sequences
- Restriction enzymes catalyse the hydrolysis of phosphodiester bonds
You end up with an overhang
Different restriction enzymes recognise different specific DNA sequences
Recognition sites (aka recognition sequences) are 4-8 base
pairs in length, depending on the enzyme, and palindromic.
Some nucleases produce an overhang… either a 5’ overhang or a 3’ overhang
…some nucleases produce a blunt end.
What does DNA ligase do?
Incorporates phosphodiester bonds between the phosphate and hydroxyl groups
Repairs nicks in phosphodiester bonds
What are the main uses of DNA polymerases?
PCR amplification
Generation of probes.
Blunt-ending of DNA overhangs to make it compatible for ligation with an already blunt ended fragment
What does a phosphatase do?
Hydrolyses a phosphate group off its substrate, so removes phosphate group
Initially we used calf intestinal alkaline phosphatase
Now we use shrimp alkaline phosphatase
What is apolynucleotide kinase?
Kinase: phosphate from ATP to substrate
ATP + SUbstrate —(kinase)—> ADP + Phosphorylated substrate
Polynucleotide kinase adds phosphate to 5’ hydroxyl 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 labelled ATP
- fluorescently labelled ATP
What are probes?
Fragment of ssDNA (or RNA)
20 – 1000 bases in length
Complementary to the gene of interest
What is reverse transcriptase?
RNA dependent DNA polymerase (uses RNA template and makes DNA copies)
Isolated from RNA-containing retroviruses e.g. HIV
Synthesizes a DNA molecule complementary to a mRNA template using dNTPs
RNA dependent DNA polymerase so needs primer, RNA template and dNTPs
How do you prime for reverse transcriptase?
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
Binds to poly A tail using a long complementary series
Gene specific primer can be used in combination with oligo(dT) primer
