gene cloning

what type of method is pcr

in vitro

what type of method is using a host cell and vector

in vivo

state the 4 steps of in vivo cloning

insertion, transformation, identification, growth

what needs to be added to the fragment before being inserted

promoter and terminator

promoter

dna sequence where transcription and rna polymerase can bind to start transcription

terminator

dna sequence which releases rna polymerase to stop transcription

another enzyme needed to add to fragment

dna ligase

role of dna ligase

to join the dna fragment and plasmid by forming phosphodiester bonds (backbone)

how is the dna fragment inserted into the plasmid

dna fragment with promoter and terminator is inserted into plasmid vector, same restriction endonuclease cuts fragment and plasmid leaving complementary sticky ends, dna ligase pairs fragment and plasmid

transformation

reintroduction of plasmids to host bacterial cells using calcium chloride or heat shock to increase the number of cells that take up recombinant plasmids

2 types of marker genes

antibiotic resistance, fluorescent

process of using antibiotic resistance genes to identify transformed bacteria

antibiotic resistance marker gene is added to plasmids, plasmids added to bacteria and grown on a plate containing antibiotic resistance (marker gene is expressed), bacteria that are not killed off likely to have plasmid and desired gene

growth

identified recombinant bacteria are cultured in a fermenter on industrial scale, products are extracted and purified, or plasmids used as source of many copies of gene for other processes

key characteristic of dna polymerase used in pcr

thermostable

pcr prep

dna fragments, thermostable dna polymerase, small primer sequences and nucleotide bases are mixed in a vial

primer

short sequences of nucleotide bases (single stranded dna) that are complementary to a particular dna sequence

basic aim of pcr

make more copies of the original dna

1st step of pcr (separate...)

dna (reaction mixture) is heated to 90-95 C for about 30 seconds, this breaks the hydrogen bonds between the dna strands, which separate

2nd step of pcr (temp...add...)

mixture is cooled to allow primers and nucleotides to bind, primers are added which bind to the dna strands to start the copying process

3rd step of pcr (temp...add...)

mixture is heated again to 75 C and dna polymerase is added, dna polymerase adds free nucleotides to primer segments and builds up complementary dna strand

final step of pcr (after 1 cycle)

cycle repeated many (around 30) times to give many copies of the original dna

2 strengths of in vitro cloning

very rapid, does not require living cells

3 strengths of in vivo cloning

very accurate, useful for transferring genes to other organisms, no contamination risk