nucleic acids and chromosomes Flashcards
nucleic acid analysis: explain the methods used to analyse nucleic acids, including hybridisation, the polymerase chain reaction (PCR) and the use of restriction enzymes
hybridisation: preparation
electrophoresis separates DNA on size; after resolution, DNA isolated from gel and moved to membrane to form replica for hybridisation
hybridisation: replica
detects specific nucleic acid sequences where homologous ssDNA/RNA combine to form a double stranded molecule
standard assay
labelled nucleic acid probe identifies homologous related target molecules (hybridised with solution of radioactive/fluorescent labelled probe); detected by photographic film exposure
hybridisation targets
DNA, RNA, chromosome
define stringency
power to distinguish between related sequences
conditions to increase stringency
increase T or decrease [Na+]
high stringency and use
duplexes are exactly complementary; used for expression profiling (e.g. identifying disease genes)
low stringency
increases chance of mismatch
how to denature probe DNA
heat until H-bonds disrupted - depends on length, bases (G-C requires greater energy than A-T), chemical environment (Na+ stabilises PO4 3-); denaturants
nucleic acid duplex stability
Tm measues nucleic acid duplex stability: midpoint temperature where double stranded transitions into single stranded
at what temperature does hybridisation occur
25C below Tm
PCR: aim
selective amplification of specific target DNA
PCR: requirements
sequence info to make 2 primers, Taq polymerase, dNTPs
PCR: primers
2 required to be complementary to each strand copied in 5’ to 3’; long enough to be specific; no tandem repeats; equal Tm; no complimentary bases at 3’ (otherwise would form dimers)
PCR: method
denature at high T; anneal at low T; extend at high T
PCR: use
point mutations, cDNA cloning, gene expression, sequencing and microarrays (microscopic cDNA spots arrayed)
restriction endonucleases: function
cut target DNA and replicon (e.g. plasmid)
restriction endonucleases: forming recombinant DNA
DNA ligase anneals; recombinant inserted into host cell; selective propagation; expansion and isolation of only recombinant DNA
restriction endonucleases: type II
cleave at specific palindromic recognition sites, producing sticky or blunt ends
relationship between recognition site length and frequency present
as recognition site length increases, frequency found in DNA decreases
