Analysis of Nucleic acids Flashcards
DNA cloning
Selective amplification of DNA sequences of interest to generate homogenous DNA populations
Can be done in vivo or in vitro
Describe in vivo cloning
- Replicon and target DNA is cut with the same restriction endonuclease (so ends are compatible)
- Mix together
- Join using DNA ligase
- Transform recombinant DNA into host cell e.g. yeast
- Selective propagation of individual colonies on agar plate- use antibiotic resistance marker so only cells that take up replicon survive
- Expansion of cell culture and isolation of recombinant DNA
Describe type II restriction endonucleases
Enzymes that cleave DNA at specific recognition sequences
Recognition sites are 4-8bp palindromic sequences
Sticky or blunt ends
What is the result of host DNA being protected by methylation of a base in the RE site by a specific methylase?
Restriction endonuclease will only cleave un-methylated DNA from invading organisms, not host DNA
Describe electrophoresis
DNA is negatively charged due to phosphate backbone
so moves to the anode (+ve electrode)
Smaller and more negatively charged fragments move further
What is electrophoresis used for?
Separation of DNA fragments
What happens after resolution in electrophoresis?
DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation
What is nucleic acid hybridisation?
method for detecting specific nucleic acid sequences
Homologous single-stranded DNA or RNA molecules combine to form double-stranded molecules
Labelling in nucleic acid hybridisation
Standard assay involves a labeled nucleic acid probe to identify homologous related target molecules in a mixture of unlabelled nucleic acids
What occurs in hybridisation assays?
Target DNA is immobilised on a solid support (e.g. nylon) which readily binds ss nucleic acid (denatured DNA/ mRNA)
Then hybridised with solution of labelled probe
Hybridisation blotting of DNA fragments after separation
After resolution, DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation with a labelled probe detected by exposure of photographic film
What is southern blotting?
Making electrophoresis fragments into immobilised hybridisation ones.
Can be washed with probes and then probes washed away
Can use photographic film to see where the bands are
Types of hybridisation assay
Southern blot: DNA target + DNA probe
Northern blot: RNA target + DNA probe
Colony blot: bacterial DNA target + DNA probe
Tissue in situ: RNA target + RNA probe
Chromosome in situ: chromosome target + DNA probe
What does reverse hybridisation involve?
Microarrays (immobilised DNA or oligonucleotide probe, target DNA solution)
What can Gene specific probes be used for?
To detect restriction fragment length polymorphisms and identify organisms genotype
What can in situ hybridisation be used for?
To locate specific genes on chromosomes
How do you denature a probe DNA?
Heat until hydrogen bonds between bases holding 2 strands are disrupted
Energy needed to denature DNA probe depends on…
Length: longer strand = more hydrogen bonds
Base composition: GC pair has 1 more hydrogen bond than AT, so harder to break
Chemical environment: monovalent cations stabilise DNA duplex by neutralising charge on phosphate backbone
denaturants destabilise DNA duplex
Define melting temperature
The midpoint temperature of transition between double stranded to single stranded nucleic acids
Melting temperature for mammalian genomic DNA and temperature hybridisation is carrier out at
Tm= 87 degrees C
Hybridisation is carried out at <25 degrees below Tm
What is hybridisation stringency?
The power to distinguish between related sequences
What does hybridisation stringency increase with?
Increase in temperature
Decrease in Na+ concentration
What occurs at high stringency?
Duplexes form only between strands with perfect 1-to-1 complementarity
What occurs at low stringency?
Annealing between strands with some degree of mismatch between bases
What is the in vitro method of DNA cloning?
Polymerase chain reaction (PCR)
Describe PCR methodology
Denature DNA (94 degrees)
Anneal DNA with primer (50-60 degrees)
Extend primer with DNA polymerase (72 degrees)
= Thermostable Taq polymerase added with dNTPs to extend 5’ to 3’ from primers to generate new stands
What to consider when designing a primer
Length: 20 nucleotides, gives required specificity
Base composition: no tandem repeats that can form hairpins
Avoid complementary at 3’ end: as primers join to each other instead (primer dimers)
What needs to be designed before PCR can be carrier out?
2 primers
1 complimentary to each strand of the DNA to be copied
Name 5 applications of PCR
Typing genetic markers Detecting point mutations DNA sequencing DNA microarrays Genome walking
What is a DNA (or oglionucleotide) microarray?
a collection of microscopic DNA (or oligonucleotide) spots, commonly representing single genes, robotically arrayed on a solid surface
What do qualitative or quantitative measurements with DNA microarrays utilise?
the selective nature of DNA-DNA or DNA-RNA hybridisation under high-stringency conditions, with fluorophore-based detection
What are DNA microarrays used for?
Used for expression profiling
To monitor expression levels of 1000s of genes simultaneously or comparative genomic hybridisation
How can microarrays be used to identify disease genes?
By comparing gene expression in diseased and normal cells
