DNA amplification and genetic engineering Flashcards Preview

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

what components are needed in PCR

-template DNA (single stranded)
-primers
-dNTPS- nucleotides
-buffers (mg2+)
-Taq polymerase

2

Uses of PCR

-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

limitations of PCR

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

PCR process

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

TAQ polymerase is..

thermostable--> extremophile

6

power of genetic engineering

-genes can be shared between species
-geentic code is universal so expression is possible

7

useful products produced using genetic engineering

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

8

gel electrophoresis

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

why is there a signifiant mutation rate in PCR

Taq pol does not have checking mechanism

10

denaturation

95

11

annealing

50

12

extension

72

13

only dna between.. is amplified

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

14

ends of the amplified frag are defined by ..

2 primers

15

after 30 cycles of PCR...

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

16

PCR primers are

short 20bp single stranded DNA
-OLIGONUCLEOTIDES

17

for expression of eukaryotic genes

normally need to make cDNA copy of mRNA

18

dna moves through gel depending

size and shape -smaller the faster due to less reissitance

19

dye used for staining

ethidium bromide

20

genetic engineering basic

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

gene cloning

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

22

reporter genes

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

in vivo cloning

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

e.g. of calculating recognition sites

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

25

what is a gene library

a collection of recombinant clones

26

what can a gene library be used for

-can screen for clones containing gene of interest

27

what is plasmid cut with and then rejoined with

restriction endonuclease
ligase--> requires ATP

28

where is cloning a very large DNA possible

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