Analysis of nucleic acids Flashcards
Describe how to construct DNA molecules in vitro
cutting a target DNA and a replicon with restriction endonucleases, so that the ends of the two DNA sequences are compatible. Joining the DNA fragments by using the enzyme DNA ligase.
Transformation of the recombinant DNA molecules into host cells (bacteria, yeast).
Selective propagation of individual cell colonies (selectable antibiotic resistance markers).
Expansion of the cell culture and isolation of recombinant DNA.
what are restriction endonucleases
Enzymes that cleave DNA at specific recognition sites, usually 4-8bp palindromic sequences – produce “blunt” or “sticky” DNA ends.
The longer the recognition site, the less frequently it occurs in DNA.
What are the ways to seperate DNA fragments
Electrophoresis – DNA is negatively charged due to its phosphate backbone and moves towards the anode (+ve electrode) when an electrical force is applied to it.
After resolution, DNA can be isolated from the gel or transferred to a membrane to form a replica for hybridisation.
what is nucleic acid hybridisation?
A key method for detecting specific nucleic acid sequences in which homologous single-stranded DNA or RNA molecules combine via homologous base-pairing to form double-stranded molecules.
what is standard assay
Standard assay involves a labeled nucleic acid probe (DNA, RNA or oligonucleotide) to identify homologous related molecules in a mixture of target unlabeled nucleic acids.
what is hybridisation assays
Target DNA is immobilised on a solid support – nylon or nitrocellulose membrane – which readily binds single-stranded nucleic acid (e.g. DNA) and then hybridised with a solution of labelled probe (radioactive or fluorescent).
e.g. Southern blot hybridisation (DNA target and DNA probe)
Northern blot hybridisation (RNA target and DNA probe)
Colony blot hybridisation (bacterial DNA target, DNA probe)
Chromosome in situ hybridisation (Chromosome target and DNA probe)
Tissue in situ hybridisation (RNA target and RNA probe)
Reverse hybridisation – Microarrays (immobilised DNA or oligonucleotide probe, target DNA solution)
how does chemical environment affect melting temperature?
chemical environment (monovalent cations stabilise the DNA duplex by neutralising charge on phosphate backbone; denaturants (formamide/urea) destabilise the DNA duplex).
what is hybridisation strigency
(i.e. the power to distinguish between related sequences) increases with increase in temperature and decrease in salt concentration.
what is PCR
In vitro method allows selective amplification of a target DNA within a heterogeneous collection of DNA sequences
denature and repeat the cycle several times to generate many copies of the target DNA.
- Denature
- Anneal
- Extend
NEED:
-2 primers (15-25 nucleotide in length), one complementary to each strand of the DNA to be copied
-Primers are specifically annealed to heat denatured DNA.
-thermostable DNA polymerase + dNTPs extend and generate from the primers and generate new strands
steps:
Denature the DNA and repeat the cycle many times -> geometric increase.
What information about nucleic acids can be revealed by the technique of Southern blotting?
Identity of DNA, size and abundance
what should the primer in the PCR ensure to have
Primer needs to avoid having tandem repeats at its ends, otherwise its 3’ and 5’ will loop and bind to each other
Primer needs to a NON-complimentary sequence to the 3’ end to ensure it binds to the 5’ end
what is DNA microarray
monitors expression levels of thousand of genes
what is melting temperature and how is this used to work out what temp to do hybridisation?
The temperature at which half of the nucleic acid duplexes have broken down into single strands.
Hybridisation is carried out at temperatures < 25oC below Tm
