Recombinant DNA Flashcards
(21 cards)
TRANSGENIC meaning
the genetic code, transcription and translation machinery is universal so transferred fragments of DNA can be translated within cells of recipient organisms
3 methods:
Reverse transcriptase
Restriction endonuclease
Gene Machine
Reverse transcriptase method
1) isolate cell which produces large amounts of desired protein
2) find large amounts of mRNA to code for that protein
3) isolate mRNA and add reverse transcriptase- complementary base pairing between mRNA and free nucleotides produce single stranded cDNA
4) DNA polymerase makes second strand of cDNA
Pros of reverse transcriptase method
no introns
Restriction endonuclease method
- enzymes with active site- complementary shape to specific base sequence on DNA ‘restriction site’
- Cut at same point on both strands of DNA, or staggered ends so bases are exposed- creates sticky ends.- palindromic
Gene machine:
1) scientists identify amino acid sequence, then mRNA sequence, the DNA sequence.
2) DNA sequence entered into a computer (biosafety and biosecurity-international standards and ethical requirements
3) computer makes ‘small sections of overlapping nucleotides- ‘oligonucleotides’
4) oligonucleotides joined to form DNA sequence of entire gene.
5) PCR multiplies gene to get multiple copies
Pros of gene machine
no introns or non coding DNA
fast, many short sequences produced in short period of time
In vivo cloning process:
1) DNA fragment cut by restriction endonuclease enzyme
2) promoter and terminator regions added to allow for RNA polymerase to bind and detach
3) plasmid cut using same endonuclease and DNA fragment inserted into plasmid
4) comp base pairing between sticky ends+ DNA ligase joins/ sticks DNA together- phosphodiester bonds
5)Transformation:
heat shock cell- sudden increase and decrease in temperature
Ca2+ increase permeability
to allow for bacteria to take up plasmids
Problems with in vivo:
recombinant plasmids don’t enter cells
DNA fragment sticks to itself
plasmid re-joins before DNA fragment joins
marker genes examples:
antibiotic resistant genes
fluorescent genes
genes coding for enzymes
Antibiotic resistance gene
1) DNA fragment inserted into antibiotic gene- no longer antibiotically resistant
2) replica planting technique: press agar onto another plate with antibiotic- the bacteria that died took up DNA
Fluorescent markers
1)DNA fragment inserted inside fluorescent gene- Bacteria that took up DNA will no longer fluoresce
enzymes:
1)lactase enzyme turns substances colourless to blue
2)insert gene in middle of lactase DNA- disrupts it
3)grow bacteria on lactase mixture- blue=did not take up DNA
colourless=took up new DNA
In vitro process:
PCR:
- Mixture- DNA polymerase, DNA nucleotides, primers, Mg2+
1) heat to 95 degrees to break hydrogen bonds to split DNA into single strands
2) temperature decreases so primers can anneal
primer: short DNA sequences that allow the attachment of DNA polymerase
3) heat to 72 degrees (optimum temperature for DNA polymerase)- DNA polymerase forms phosphodiester bonds between nucleotides by condensation reaction
advantages of PCR:
1) automated
2) rapid
3) doesn’t require living cells
4) can produce large quantities of product
In vivo cloning advantages:
Useful when you want to introduce a gene to another organism
no risk of contamination- same endonuclease
it is very accurate
can produce large quantities of product
What are DNA probes?
what is this used for?
short single stranded length of DNA that is labelled so it can be easily identified- examples of labels include radioactively or fluorescently labelled
locate specific alleles of genes and screen patients
DNA hybridisation process:
looking for a specific allele in a patient:
1) determine base sequence of desired allele using genetic libraries
2) fragment of DNA produced for desired allele
2) make DNA probe by attaching marker to it and then replicate DNA probe using PCR
3) heat patients DNA to separate into 2 strands by breaking hydrogen bonds
4) cool in a mixture containing DNA probes, if desired allele present, probes and DNA will bind
5) wash to remove unbound probes
6) shine light on fragment to cause fluorescence
What does screening allow for
allows for doctors to select medicines based on your genotype- safer and more cost effective
genetic counselling- giving advice and information to how people can change their lifestyles
What are VNTRs and how are they used for genetic fingerprinting?
1)VNTRs -non coding base sequences of DNA contained in introns- probability of two individuals having the same VNTRs is low unless related
gel electrophoresis process:
1) cut DNA using restriction endonuclease
2) use electrophoresis - separates by mass/ length
3) transfer to nylon sheet
4) add alkali- separates DNA into 2 strands
5) add probes
6) replicate using PCR
7) test for probe and compare vnts
