DNA amplification and genetic engineering Flashcards Preview

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Flashcards in DNA amplification and genetic engineering Deck (28)
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1
Q

what components are needed in PCR

A
  • template DNA (single stranded)
  • primers
  • dNTPS- nucleotides
  • buffers (mg2+)
  • Taq polymerase
2
Q

Uses of PCR

A

-amplification od DNA
-sequencing
-detection of pathogens in water or blood
-genetic fingerprinting
forensic analysis
-diagnosis of genetic disorders
-prenatal diagnosis
-analysis of ancient DNA

3
Q

limitations of PCR

A
  1. two specific primers needs to be made
  2. limit on length of amplified frag
  3. high mutation rate- taq does not have a proof reading mechanism
  4. sensitive to exact reaction conditions
  5. tiny amounts of contaminating DNA will also be amplified
4
Q

PCR process

A

carried out in a thermocycler- repeats itself 30-40 times

  1. denturaton- 95 degrees- DNA becomes single stranded
  2. annealing- 50 degrees. primers bind to dna and polymerase attaches and starts the synthesis of DNA
  3. extension- 72 degrees- optimum temp for TAQ pol- extension fragment
5
Q

TAQ polymerase is..

A

thermostable–> extremophile

6
Q

power of genetic engineering

A
  • genes can be shared between species

- geentic code is universal so expression is possible

7
Q

useful products produced using genetic engineering

A
human insulin
blood clotting factor
human growth hormone
bovine chymosin
hepatitis B vaccine
artemisini (anti-malarial)
8
Q

gel electrophoresis

A

fractionation of dna depending on size

  • potential difference applied along a gel
  • dna moves to +ve electrode through gel depending on: conformation (shape) and size
  • dna is stained with fluorescent dye for detection by UV exposure e.g. ethidium bride
  • moves towards positive end due to negative phosphate group1
9
Q

why is there a signifiant mutation rate in PCR

A

Taq pol does not have checking mechanism

10
Q

denaturation

A

95

11
Q

annealing

A

50

12
Q

extension

A

72

13
Q

only dna between.. is amplified

A

primers- specific sequences can be amplified from a complex mixture of dna

14
Q

ends of the amplified frag are defined by ..

A

2 primers

15
Q

after 30 cycles of PCR…

A

10^6 fold amplification–> enough for analysis on gel

16
Q

PCR primers are

A

short 20bp single stranded DNA

-OLIGONUCLEOTIDES

17
Q

for expression of eukaryotic genes

A

normally need to make cDNA copy of mRNA

18
Q

dna moves through gel depending

A

size and shape -smaller the faster due to less reissitance

19
Q

dye used for staining

A

ethidium bromide

20
Q

genetic engineering basic

A

involves the introduction of foreign in DNA into cells. The the cell will replicate and express the dna e.g. in plasmids by inserting a transformed plasmid

21
Q

gene cloning

A
  • introduce rDNA (recombinant plasmids0 into bacterial cell
  • will replicate when cells divide
  • dna amplified
  • recover dna for analysis (purify)
22
Q

reporter genes

A

proteins tagged with green fluorescent protein (GFP)
-from bioluminescent jellyfish
-autcatalytic, fluorescent protein
0in vivo reporter, high signal to to noise, no enzyme activity
-many spectral mutant available.

23
Q

in vivo cloning

A

insertion of dna into bacteria
-plasmid is cut with a restriction endonuclease (bacterias defence mechanism)–> specific enzymes t a specific recognition sequence
-enzyme will leave staggered cut–> by cleaving phosphodietser linkages
DNA ligase will jin the DNA together by reforming phosphodieter bonds (requires ATP)

24
Q

e.g. of calculating recognition sites

A

sequence occurs every 1/5096bp–> 4.6x10^6/4x10^3=1150 sites

25
Q

what is a gene library

A

a collection of recombinant clones

26
Q

what can a gene library be used for

A

-can screen for clones containing gene of interest

27
Q

what is plasmid cut with and then rejoined with

A

restriction endonuclease

ligase–> requires ATP

28
Q

where is cloning a very large DNA possible

A

yeast artificial chromosomes (YACs)
e.g. artemisinin production- produced in yeast- treatment against malaria–> originally found in plant, however gene recovered and inserted into yeast, grows much more effectively (by feb 2013 40 million treatments produced