Nucleic acid methods Flashcards
How can DNA be used in experiments?
Restriction enzymes
Cloning
PCR
DNA gels
How can RNA be used in experiments?
QPCR or RTPCR
RNA sequencing
RNA-ish
How can proteins be used in experiments?
Western blotting
Immunohistochemistry
Immunofluorescence
Flow cytometry
How is DNA used in the lab?
For basic understanding of genomes - by sequencing and mapping.
Manipulating DNA - changing the genome, adding additional genetic material.
Why is DNA changed?
Gene therapy - correct copies of faulty genes in host organism
Genetically modified organisms
Sequencing - fragments of genomic DNA inserted into different cloning vectors to allow short lengths of sequencing
What is recombinant DNA technology?
Experimental methods leading to the transfer of genetic information from one organism to another.
Need to make lots of copies of the original DNA - gene cloning.
What is a clone?
A collection of molecules or cells, identical to an original molecule or cell.
What is gene cloning?
Making copies of a gene.
Can be a normal copy - wild type.
Can be an altered version of a gene - mutant.
How is genetic material sourced for cloning?
Cut the gene out of DNA
Restriction endonucleases from bacteria, cleave double stranded DNA in a sequence-specific manner.
What are restriction enzymes?
Evolved as a defence mechanism in bacteria against infection by foreign DNA.
Different restriction enzymes are in different bacteria.
Protect the host DNA by methylation.
What are type II restriction enzymes?
Cut palindromic sequences - double stranded sequences where 5’-3’ is the same as 3’-5’.
What is the restriction site?
Each enzyme recognises a small base pair sequence.
The enzyme then cuts (digests) the DNA at a specific sequence - the restriction site.
How do type II restriction enzymes cut?
Cleavage can leave staggered or sticky ends
Can be 3’ or 5’ overhangs
Or produce blunt ends
What are sticky ends?
5’ or 3’ overhangs.
Enzymes with staggered cuts have complementary ends.
What are blunt ends?
Enzymes cut at the same position on both strands to leave blunt ends.
So the plasmid does not need to match, but it can be hard to replicate the same cut.
What is DNA ligase?
Can join complementary strands.
These are cut with staggered cuts to be ligated together.
Sticky ends that are not complementary cannot be ligated together.
How does DNA ligase join blunt ends?
DNA fragments with blunt ends generated by different enzymes can be ligated together with lower efficiency.
They are not usually re-cut by the original restriction enzyme.
But if they constitute a Smal or Dral site - CCCGGG, AAATTT they can be cut by Smal or Dral.
What are cloning vectors?
Plasmids, naturally occurring extrachromosomal DNA molecules.
They are small circular double stranded DNA molecules.
Can be modified to carry new genes.
Can also replicate DNA.
What are the requirements of plasmids as cloning vectors?
Origin of replication - to replicate themselves.
Selectable marker - e.g. resistance gene, to see that the bacteria has the plasmid in it.
Multiple cloning sites - where insertion of DNA will not disrupt replication or inactivate essential markers.
How can genes be used to select for plasmids?
LacZ gene has multiple cloning site, when intact it lets bacteria break down galactose.
Media can make bacteria blue if they have this gene.
If a gene is put inside the cloning site, colonies will be white, no LacZ.
How is a gene cloned into a plasmid?
Use the same restriction enzyme to cut the plasmid and genomic DNA.
Ligate the DNA fragment with the gene into the plasmid - they have compatible ends.
Transform into E.coli to grow.
Select for cells with plasmid on an agar plate with antibiotic.
How can a plasmid enter the human cells?
Transfect into competent bacterial cells.
Or human cell line - bacteria make proteins different, so need to check the drug has the right structure.
Electrocute cells to have holes, take up DNA.
Use CaCl2.
Use lipofectin, dissolves the cell membrane.
Select it, grow in culture, or human cells in a flask.
What is genetically modifying genomic DNA?
Changes to gDNA in stem cells:
Stop genes functioning
Put in a reporter gene.
Deliberately mutate a gene to model a human disease.
What is Cas9?
A bacterial nuclease that defends against double stranded DNA viruses.
It uses guide RNA to create double stranded breaks in the DNA - creates precise cuts.
Cellular DNA repair mechanisms trim back the DNA.
Normally the breaks are repaired by homologous DNA repair.
What is CRISPR/Cas9 gene editing?
Use the Cas9 to make precise cuts.
Then provide the DNA to be used in the repair to change the gene as desired.
