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
