Define recombinant DNA technology
the transfer of DNA fragments from one organism to another
Give facts about Recombinant DNA Technology
- universal genetic code i.e. transcription/translation mostly similar in organisms
- transferred genes can code for proteins in the recipient
- transgenic organisms (contain transferred DNA)
How can genes be isolated and copied using reverse transcriptase enzymes?
- isolate cells
- extract mRNA and add reverse transcriptase + free DNA nucleotides to form a single stranded complimentary copy of DNA (cDNA) from an RNA template
- add DNA polymerase to make double stranded DNA
whats the advantage of the reverse transcriptase method?
- mRNA doesn’t contain introns
- mRNA present in large amounts making protein
How can genes be isolated and removed using restriction endonuclease enzymes?
- RE recognise and cut recognition sequences which are palindromic DNA sequences
- shape of active site complementary to recognition sequence
- leaves sticky ends which can easily bind/anneal to any other DNA fragment cut using the same restriction enzyme
define sticky ends
small tails of unpaired bases
define oligonucleotides
short length of DNA about 20 nucleotides
How can genes be synthesised from scratch using a ‘gene machine’
- design new sequence to make desired amino acid sequence of the new protein
- attach the first nucleotide to a bead and add others step by step
- add protecting groups to ensure nucleotides bond in correct position
whats the advantage of a gene machine?
faster to use compared to all the enyzme-catalysed reactions
Whats the benefits of DNA amplification in vitro by PCR?
- large no. of copies of any specific DNA fragment produced
- enables test to be carried out on small/old samples
- repeat 25 times to get millions of DNA copies
Explain the process of DNA amplification in vitro by PCR
- Denaturation-heat to 95 degrees to break H bonds separating DNA strands becoming templates for new complimentary strands
- Annealing-cool to 55 degrees means primers attach to DNA template strands at complimentary sequence as H bonds reform
- Synthesis of new DNA-raise to 70 degrees as optimum for DNA polymerase which attaches to primer and adds new bases complementary to template
Explain DNA amplication in vivo by transforming host cells
- isolate gene and cut DNA using restriction endonuclease cutting at specific base sequences to produce fragments with sticky ends
- insert gene into vector (needs to have promoter/terminator region) and plasmid DNA cut using same restriction endonuclease to produce same sticky ends so gene fits in as unpaired bases complimentary.DNA ligase anneals donor and vector DNA=recombinant DNA
- uptake of new plasmids use marker genes as identification of uptake of microorganisms
- culture E.coli produces many clones with copies of gene which produces lot of protein which is separated and purified.
