Analysis of Nucleic Acids Flashcards
Describe the creation of recombinant DNA molecules in In vivo cell-based cloning
Target DNA and a Replicon cut with the same restriction endonuclease. Mixed and then DNA ligase added
What is a replicon?
Sequence capable of independent replication
Name 3 Replicons
Plasmin, Lambda phage, Yeast artificial chromosome
What is done with Recombinant DNA in cell-based cloning?
Transformed into a host cell e.g. Yeast/bacteria, which will replicate the replicon and itself
How is a host cell containing recombinant DNA selected and replicated in cell-based cloning?
The host cell is placed on a agar plate with an antibiotic, so only the cells containing target DNA and Antimicrobial resistance gene can survive.
Colonies transfered to growth medium to replicate
Why is the same restriction endonuclease for both target DNA and the replicon in cell-based cloning?
So the two nucleotides are compatible with each other
What is a restriction endonuclease?
Enzyme that cleaves DNA at specific 4-8bp palindromic sequence to introduce blunt/sticky ends
What does gel electrophoresis do?
Separates DNA fragments based on size
How does gel electrophoresis work?
DNA is negatively charged due to phosphate backbone.
When fragments placed in solution and current is applied, they travel towards positive electrode
Which DNA fragments travel further in gel electrophoresis?
Smaller fragments as they are less hindered by mass
What is Nucleic acid hybridisation?
Method of detection of specific sequences using labelled probes
How can fragments from electrophoresis be identified?
Transfer to hybridisation assay
What is a hybridisation assay
Target DNA immobilised on solid support which readily binds single stranded nucleic acids, then probe DNA added in solution over membrane. Denaturing then allows the probe dsDNA to become ssDNA, then allowing the probe to bind to any target DNA if it is present, and any excess washed away
How can probes be detected?
Can be radioactively or fluorescently labelled
What are the targets and probes in:
Northern Blot
Southern Blot
Northern: RNA target + DNA probe
Southern: DNA target + DNA probe
What are the targets and probes in the following assays:
Colony Blot
Tissue in situ
Colony: Bacterial DNA target + DNA probe
Tissue: RNA target + RNA probe
What are the targets and probes in:
Chromosome in situ
Reverse hybridisation (microarray)
Chromosome: Chromosome target + DNA probe
Microarray: immobilised DNA or oligonucleotide probe + target DNA solution
How would you perform southern blotting ?
After electrophoresis carried out
Fragments immobilised on nylon filter and radio probes added to bind to comp DNA. X-ray film used to detect probe position
What is the melting temperature of DNA at 40% GC?
87 oC
Why might the Tm of DNA be higher?
If it contained higher % GC, as more H bonds to break
What is hybridisation stringency?
The power to distinguish between related sequences
What happens at high hybridisation stringency?
Duplexes only form between perfectly complimentary strands
How might lower hybridisation stringency be encouraged?
Lower temp, higher Na+ conc
What is required for PCR?
2 Primers Target DNA Taq polymerase dNTP's (Heat)
How is PCR carried out?
- DNA denatured at 95oC
- Annealing at 50-60oC with dNTPs by Taq poly
- extension at 72oC
- Repeat many times in thermocycler
How long is a PCR primer and why?
~20bp
Long enough to be specific to target.
How is a PCR primer designed?
Avoiding tandem repeats to prevent hairpin formation.
%GC standardised to give roughly equal Tm for each primer.
Complementarity of 3’ ends avoided to prevent primer dimerisation.
Why are 2 primers required for PCR?
One for each strand of DNA
foreward and reverse
How can a microarray be designed to show abnormal gene expression?
RNA solution + DNA probe
Cancer cells probe stained red
Normal cells probe stained Green.
Placed on same microarray, to show colour combinations.
