manipulation of DNA Flashcards
what’s so special about restriction enzymes? what are palindromes?
they recognise specific nucleotide sequences;
palindrome: sequence reads the same on both strands
how is DNA visualised?
add ethidium bromide to agarose gel-> binds to DNA-> fluorescence under UV light
why are plasmids used in cloning DNA molecules
plasmids have an origin of replication-> replication independently of chromosome-> many plasmids in each bacteria;
plasmids also have antibiotic resistance genes-> selective bacterial growth
how are plasmids used in cloning DNA molecules
restriction enzyme cuts plasmid + DNA fragment-> same complementary ends-> mixed together-> anneal to stick ends-> DNA ligase bonds them-> recombinant DNA molecule
how is cloned DNA amplified?
yeast/ E coli grown in ampicillin containing medium-> transformation:take up recombinant plasmids at an incidence of 0.1%-> cells which do not take up recombinant plasmids (contain antibiotic resistance gene) die -> only cells with recombinant plasmids survive-> DNA replication
how are human eukaryotic genes cloned ? y liddat
use cDNA (cDNA is a DNA copy of mRNA, produced by reverse transcriptase) normal DNA isnt used because it has introns
human insulin production
insulin producing gene + bacterial plasmid cut with same restriction enzyme-> mixed tgt-> recombinant DNA-> plasmid inserted into bacterium-> bacterium multiplies and produces human insulin-> insulin extracted and purified
how is DNA polymerase stopped
use dideoxy nucleotides (they have a H instead of OH at 3’ carbon-> phosphodiester bond cant be formed
how does automated DNA sequencing take place
use all 4 ddNTP, each labelled with fluorescent marker, each marker has different marker that is excited by laser light
sequences separated on capillary gel
smaller fragments move faster
how does NGS work
short reads of nucleotides on microchips-> align contiguous sequences
what kind of PCR polymerases are used in PCR
thermostable: taq/pfu
PCR steps; number of copies produced after x cycles
denaturation: 95 deg– denature dna into single strands
primer annealing: cool to 48-65– primers anneal to complementary sequence
primer extension: 72 deg– taq polymerase synthesises dna
2^x
where do PCR reactions take place?
thermocycler
how do DNA microarrays work
mRNA-> cDNA-> fluorescently labelled-> denatured into single strands-> used as probe-> applied to array which contains spots of ssDNA(oligonucleotides: short dna molecules) from each gene -> cDNA binds to spots that have complementary sequences-> fluorescence (intensity proportional to expression level)
