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 2) using enzymes

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

different methods used 3) 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

DNA probes

-short single-stranded DNA that is complementary to the base sequence of the target gene

-have radioactive/fluorescent label attached for detection

-used after electrophoresis as DNA is invisible on gel so allows detection

DNA microarray

-screens lots of of different alleles at the same time

-probe used to check if sample of labelled DNA will stick if complementary

uses of DNA probes

-to screen someone’s DNA for heritable conditions - use in genetic counselling or personalised medicines

-to determine patient’s response to drug

-to identify any health risks

DNA hybridisation

-measures degree of difference between 2 strands of DNA

process: 1 DNA labelled and mixed with unlabelled comparison strand, the more similar the strands, the more strongly they will bind so more energy needed to break strands apart

genetic fingerprinting

technique used to compare 2 DNA samples + determine whether they came from the same individual

Variable Number Tandem Repeats

-non-coding base sequences - the probability of individuals having same VNTRs is low so used to compare DNA

genetic fingerprinting PROCESS

1) DNA extracted from blood, hair, saliva, etc, then PCR used to amplify number of DNA fragments

2) then DNA cut by restriction endonucleases at specific sequences on ends into segments

3) then DNA mixture placed into well on gel with electric current passing through

4) electrophoresis = DNA fragments move towards anode + are separated according to mass or charge - smaller fragments move further than long fragments

5) then transfer pattern to nylon sheet/membrane + is fixed to it

6) then apply probe which bind by hybridisation

7) areas with probe are identified using X-ray film

8) compared to DNA ladder - each banding pattern unique to each individual

uses of genetic fingerprinting

-forensic science

-paternity cases

-medical diagnosis

-animal + plant breeding