3.8.4 Gene Technology

recombinant DNA technology

the transfer of fragments of DNA from one organism/species to another

different methods used 1) using reverse transcriptase

-mRNA is complementary to target gene so is used as a template

-mRNA mixed with free DNA nucleotides which match up to base pairs + reverse transcriptase which forms sugar-phosphate backbone to create cDNA

-cDNA is single stranded so DNA polymerase makes it double stranded

different methods used 1) using enzymes

-restriction endonucleases that recognise specific palindromic sequences + cut DNA here

different methods used 1) using a gene machine

-need to determine sequence of nucleotide bases that code for desired protein first

-oligonucleotides produced then broken off support + protecting groups which are then joined together to make longer fragments

amplifying DNA fragments 1) In vivo cloning

-vector carrying DNA plasmid is isolated

-vector cut open by restriction endonuclease which creates sticky ends so vector + DNA fragment with gene are complementary

-DNA ligase joins sticky ends together → forms recombinant DNA

-vector with recombinant DNA transferred into host bacterial cell so is transformed → cells placed in ice-cold CaCl2 solution to make walls more permeable then heat-shocked

-marker genes = used to identify transformed cells e.g. fluorescence, antibiotic resistance

amplifying DNA fragments 2) In vitro cloning

Polymerase Chain Reaction

-used to amplify small amounts of DNA obtained so enough DNA available for genetic fingerprinting

-mixture contains DNA sample, free nucleotides, primers + DNA polymerase

PCR process

-DNA mixture heated to 95C to break hydrogen bonds + separate strands

-primers + nucleotides added

-then mixture cooled so primers can bind to DNA strands - these initiate replication + keep strands separate

-then mixture heated to 72C to activate DNA/Taq polymerase which joins DNA nucleotides forming 2 new copies of DNA

-new DNA acts as a template for next cycle

primers + Taq polymerase

primer = short fragments of single-stranded DNA

Taq polymerase = is thermostable - will not denature so can withstand high temperatures