8- Enzyme and Restriction Mapping Flashcards
what is a restriction enzyme?
a protein isolated from bacteria - cleaves DNA sequences at sequence-specific sites, producing DNA fragments with a known sequence at each end
where are restriction enzymes derived from?
bacteria and archaea
natural function of restriction enzymes?
defend bacteria and archaea against viral infections
- bacterial cell is infected = restriction enzyme injects its DNA into the host cell for replication
- restriction enzymes recognise specific DNSA sequences - recognition sites - and cleave foreign viral DNA at these sites
- prevents microbe from successfully replicating inside the bacterial cell
use of restriction enzymes in molecular biology?
tools in genetic engineering and recombinant DNA technology - for cutting and manipulating DNA molecules precisely
examples of restriction enzymes?
nucleases
ligase
polymerases
phosphatase
(polynucleotide) kinase
applications for restriction enzymes in molecular diagnostics
- DNA fragmentation (PCR/ DNA sequencing)
- cleave genomic DNA at specific recognition sites = produce DNA fragments
- for sequencing, makes the fragments a more manageable size
- for PCR, allows targeted amplification of specific genomic region - recombinant DNA technology
- cut the gene of interest and plasmid vector in DNA cloning
- enables insertion of gene into vector, creates a recombinant DNA molecule - methylation specific assays, forensic analysis to generate unique DNA profiles for crime scenes and paternity testing
- genome mapping = identify location of genes, regulatory regions, other genomic features
- plasmid characterisation = understanding plasmid structure, mapping out the position of restriction enzyme recognition sites using gel electrophoresis
describe recombinant human-plasmid hybridisation
DNA molecules form different sources can be joined together - restriction enzymes are used to create complementary overhands on the human gene and plasmid vector = allows the to fuse
catalysed by DNA ligase, results in recombinant DNA molecule
how are restriction enzymes used in restriction mapping of plasmids?
plasmids are digested with restriction enzymes - different restriction enzymes cleave at their specific recognition sites producing DNA fragments of varying sizes
analyse the resulting fragments using gel electrophoresis - can determine recognition sites for plasmid restriction mapping
what is a nuclease?
enzyme that degrades nucleic acids by hydrolysing phosphodiester bonds
different types:
- ribonuclease
- deoxyribonuclease = endonuclease (cleaves within nucleotide chain) and exonuclease (degrades from end of molecule)
how do restriction endonucleases work?
endonucleases cleave within the nucleotide chain
different restriction endonucleases recognise specific palindromic recognition sites, cleave by hydrolysing phosphodiester bonds - can produce overhangs or blunt ends
list the other enzymes involved in creating recombinant DNA
DNA ligase
DNA polymerase
phosphatases
polynucleotide kinase
reverse transcriptase
how is DNA polymerase involved in creating recombinant DNA?
synthesises DNA in the 5’-3’ direction, needs a primer to polymerise nucleotides
DNA polymerase II recognises the primer, incorporates nucleotides for complementary base pairing
if two DNA fragments aren’t compatible - don’t have complementary overhangs or one has a blunt end - the fragment with a 5’ overhang can used as a primer and extended until it forms a complementary blunt-end to the other fragment
how is DNA ligase involved in creating recombinant DNA?
joins DNA molecules together to form recombinant DNA - complementary DNA fragment endings (overhangs or blunt-ends) joined
uses: gene cloning, DNA repair and replication
with restriction enzymes, DNA ligase is used to isolate and insert a GOI fragment into a bacterial plasmid
how are phosphatases involved in creating recombinant DNA?
hydrolyses and removes phosphate groups from substrates- digests DNA templates with restriction enzymes, and the DNA phosphate is removed
used to use calf-intestinal alkaline phosphatase, now we use shrimp alkaline phosphatase
prevents resealing of plasmids during cloning
how are phosphatases involved in creating recombinant DNA?
phosphate group added to the 5’ OH end of a DNA molecule - allows ligation of fragment to another, or fluorescent labelling
