making dna fragments

3 basic methods of gene isolation

conversion of mrna to complementary dna using reverse transcriptase enzymes, using restriction enonuclease enzymes to cut a fragment containing desired gene, making the gene in the gene machine

pro and con of reverse transcription

uses mrna so no introns need to be spliced out of fragments, have to have specific cell

3 basic methods of gene isolation

conversion of mrna to complementary dna using reverse transcriptase enzymes, using restriction enonuclease enzymes to cut a fragment containing desired gene, making the gene in the gene machine

pro and con of reverse transcription

uses mrna so no introns need to be spliced out of fragments, have to have specific cell

pro and con of restriction endonucleases

uses dna so can use any cell, contains introns in fragments as uses prokaryotic cells with no mechanism for splicing

pro and con of gene machine

specifically made to not contain introns, maybe more time consuming

making fragments using reverse transcription

uses mechanism of reverse transcriptase from retro visuses (makes dna from rna), mrna from cells producing desired product is obtained and reverse transcriptase produced complementary dna (cdna)

reverse transcriptase enzyme example function in insulin gene

insulin mrna extracted from b cells, reverse transcriptase makes dna, single strand of cdna is isolated, dna polymerase converts to double stranded dna

main function of restriction endonucleases

cut up dna

what are palindromic sequences

base sequences (pairs) which read the same in opposite directions (on either strand)

consequence of having introns in fragment

wrong protein produced

how do restriction endonucleases work

recognise and cut dna at palindromic sequences, producing either blunt or sticky ends, therefore if there is a suitable palindromic sequence at either side of the required fragment, dna is incubated with specific enzyme for that sequence and fragment is cut out via hydrolysis reaction

blunt ends

dna cut straight across

sticky ends

dna cut in staggered manner

why are sticky ends useful

lead tail of unpaired bases so can bind dna fragments to other complementary dna sequences e.g. on plasmid

olingonucleotides

short overlapping sections of dna around 20 bases long

gene machine basic idea (not process)

dna can be synthesised from scratch using a database

gene machine process

desired sequence worked out or designed, checked for biosafety standards, olingonucleotides formed as nucleotides are assembled, protecting groups are added to prevent unecessary branching (removed after process), multiple olingonucleotides joined to form longer dna fragment