Molecular Biology 3 Flashcards
what are mutations?
permanent alteration in a DNA (gene) sequence
what causes mutations?
errors in DNA synthesis that can occur spontaneously at low frequency, chemical mutagens, ionising radiation
what are the different types of substitution mutations?
conservative, non-conservative and no mutation
what is a conservative mutation?
amino acid is replaced by one with similar properties
what is a non-conservation mutation?
amino acid is replaced by one with different properties
what is no mutations?
a change in the third position of the codon does not change an amino acid
what is the effect of an insertion/deletion?
causes a frame-shift which synthesises a protein that has a completely different sequences downstream of the mutation
what does a stop codon do?
premature or delayed termination
what is sickle cell anaemia caused by?
single-base substitution in the beta-chain of haemoglobin
what is the substitution in sickle cell anaemia?
glutamic acid is replaced with valine
what does gene cloning produce?
large numbers of copies of a particular piece of DNA
how are genes usually cloned?
by isolating them using restriction enzymes, gel electrophoresis and then inserting into a plasmid
what do restriction enzymes do?
cut double-stranded DNA at specific DNA sequences
what is good about sticky ends
they allow DNA fragments to re-associate by base pairing
what is gel electrophoresis used to do?
separate DNA fragments on basis of their size
what is step 1 of gene cloning?
restriction enzyme is chosen that cuts on either side of the gene but not in the middle
what is step 2 of gene cloning
the gene is separated from other DNA fragments by gel electrophoresis
what is step 3 of gene cloning
suitable plasmid is linearised (cut at one point) using the same restriction enzyme
what is step 4 of gene cloning
the cut plasmid and gene are mixed, and the sticky ends of the plasmid and gene are allowed to anneal
what is step 5 of gene cloning
the annealed ends are covalently joined using DNA ligase
what is step 6 of gene cloning
the plasmid, now containing the gene of interest, is introduced into the host bacterium
what is step 7 of gene cloning?
the bacteria are grown into a colony, using antibiotic resistance genes in the plasmid to select colonies containing plasmids
what is step 8 of gene cloning
cloned cells are lysed and the plasmids isolated by centrifugation
what is step 9 of gene cloning
plasmids are cut with the restriction enzyme, releasing the cloned gene
what is DNA sequencing used for?
used to determine base sequences of DNA
what does DNA sequencing do?
work out the structure of a gene or an entire genome
what are the reaction components of DNA sequencing?
single-stranded DNA template, primer, deoxynucleotides, dideoxynucleotides, DNA polymerase, label
what is step 1 of DNA sequencing
DNA to be sequenced is mixed with primer
what is step 2 of DNA sequencing
primer binds to 3’ end of DNA
what is step 3 of DNA sequencing
DNA primer mecture divided into 2 separate reaction tubes
what is step 4 of DNA sequencing
chain synthesis proceeds in each of the four reaction mixtures
what is step 5 in DNA sequencing
gel electrophoresis separation of reaction products - band corresponding to each position of chain termination appears
what is step 6 of DNA sequencing
DNA bands detected by autoradiography or by laser in an automated sequencer
what is step 7 of DNA sequencing
DNA sequence can be deduced from the pattern of bands in the four lanes
