In Vivo/In Vitro Cloning A2 Flashcards

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1
Q

how to isolate specific genes from human DNA

A

use restriction endonuclease

to cut DNA at specific base sequence

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2
Q

define recombinant DNA

A

contains genes from two species or types of organism

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3
Q

what does reverse transcriptase do

A

makes cDNA from RNA

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4
Q

what is restriction endonuclease

A

enzyme that cuts DNA into smaller fragments
cuts at specific base sequence
breaks phosphodiester bonds

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5
Q

what is the purpose of sticky ends

A

to join 2 pieces of DNA by complementary base-pairing/binding

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6
Q

process of making recombinant DNA

A

cut desired gene from another organism
using restriction endonuclease to make sticky ends
use the same enzyme to cut plasmid
mix together and use DNA ligase to join wanted gene to plasmid

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7
Q

how to put a plasmid in a bacterial cell

A

place in solution of calcium ions to increase permeability of membrane, allowing plasmid to be taken up

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8
Q

how to identify bacteria with recombinant DNA

A

plasmids contain gene for fluorescence

plasmids contain antibiotic resistant gene which acts as marker gene

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9
Q

why is it good to use a fluorescent marker gene

A

allows quick identification of transformed bacteria under UV light

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10
Q

define vector

A

carrier of DNA into another organism

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11
Q

example of a vector

A

plasmid

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12
Q

what is a DNA probe

A

single stranded DNA
base sequence complementary to DNA that is being identified
radioactively/fluorescently labelled so it can be detected

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13
Q

define in vitro cloning

A

polymerase chain reaction

used to make many copies of gene or amplify small amount of DNA

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14
Q

temperature changes in polymerase chain reaction

A

95
52
72

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15
Q

why is PCR first heated to 95 degrees

A

H bonds break between double stranded so polynucleotides strands separate

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16
Q

why is PCR cooled to 52 degrees

A

allows primers to join DNA and add nucleotides

17
Q

why is PCR reheated to 72 degrees

A

DNA polymerase can add complementary nucleotides along each separated DNA strand

18
Q

where is the DNA polymerase used in PCR found

A

in bacteria which live in volcanic vents

19
Q

what is the purpose of primers

A

marks region of DNA to be copied

enzyme needs starting strand onto which it can attach nucleotides