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Applied Genetics > Cloning of DNA > Flashcards

Cloning of DNA Flashcards

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1
Q

What is DNA cloning

A

technique involving insertion of a foreign DNA fragment into a vector capable of replicating separately in a host cell

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2
Q

what allows multiple copies of identical DNA insertion

A

Growing the host cell allows the production of multiple copies of identical inserted DNA for use in a variety of purposes

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3
Q

what is the host cell

A

a cell that has been introduced with foreign DNA or RNA and carries it around

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4
Q

examples of methods used for DNA cloning

A
  • cloning by restriction enzyme digestion and ligation of compatible ends
  • T- A cloning directly from a PCR product
  • TOPO- attached unidirectional cloning.
  • recombination based cloning
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5
Q

what is T-A cloning directly from a PCR product

A

does not require restriction enzymes
add Adenine by PCR to 3’ ends
it relies on the ability of Adenine and Thymine on different DNA fragments to hybridize; the ligase binds them together

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6
Q

what happens to the vector in T-A cloning

A

vector linearized to make blunt ends, ddTTP added to 3’ ends

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7
Q

what is TOPO cloning

A

Vaccinia virus topoisomerase I recognises the DNA sequence 5’ –(C/T) CCTT-3’ in supercoiled DNA during replication
enzyme digests, unwinds and re-ligates the DNA at the 3’ phosphate group of the T base

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8
Q

what is TOPO - attached unidirectional cloning

A
  1. design PCR primer with CACC at its 5’ end (no modification of 3’ Primer is necessary)
  2. amplify with proofreading polymerase
  3. mix PCR product with vector, incubate 5 min, and transform E. coli
    form intermediate construct and extra sequence cleaved off in E.coli, form directionally cloned PCR product
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9
Q

what is recombination based cloning

A

takes advantage of the site-specific recombination reactions enabling the bacteriophage λ to integrate and excise itself in and out of a bacterial chromosome

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10
Q

what are the requirements for DNA cloning

A
  • foreign DNA
  • host organism
  • vector DNA for cloning
  • means of inserting foreign DNA into the vector
  • method of placing the in vitro modified DNA into the host cell
  • methods for selecting and/or screening cells that carry the inserted foreign DNA
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11
Q

what must the foreign DNA be like for DNA cloning

A

PCR product
genomic DNA
complementary DNA (cDNA)

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12
Q

what does the host organism require

A

Bacterial host – E. coli
Eukaryotic host – yeast, mammalian or plant cells
other hosts – insect cells, etc

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13
Q

what is complementary DNA

A

DNA synthesized from an mRNA by reverse transcriptase and DNA polymerase

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14
Q

what is dsDNA

A

double strand DNA

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15
Q

what is ssDNA

A

single strand DNA

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16
Q

what is a vector DNA

A

DNA molecule that functions as a “molecular carrier” that brings the DNA of interest into the host cell & facilitates its replication

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17
Q

what are the requirements for a ligation reaction

A
  • two or more fragments of DNA (blunt/cohesive)
  • buffer containing ATP
  • T4 DNA ligase
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18
Q

how is the DNA inserted into the vector to be cloned

A

foreign DNA must be ligated into the linearized vector

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19
Q

what is transference of DNA into the host cell

A

method of placing the in vitro modified DNA into the host cell

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20
Q

what occurs in a transformation event

A
  • bacterial cells take up naked DNA molecules
  • cells need to be made “competent”
  • cells are heat-shocked
  • shock allows the molecules to get inside cells
  • good efficiency of transformation ranges between 107 to 108 colonies/μg DNA
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21
Q

what is another method for DNA transfer

A

Electroporating the DNA into the host cell

22
Q

what occurs in electroporating

A
  • membranes of cells are permeablized using an electric field
  • DNA present can penetrate the membranes after the electric shock
  • protocols differ for various species
  • efficiencies of transformation ranges from 109 per μg DNA (3 kb) to 106 (136 kb)
23
Q

what is conjugation

A
  • natural transmission from donor to recipient
  • occurs with a host cell that is not readily transformed
  • formation of cell to cell junctions
24
Q

what is transfection of DNA

A
  • DNA is packaged in vitro into phage particles
  • phages are allowed to infect bacterial cells
  • DNA is transiently expressed
25
Q

what occurs in selecting and screening recombinants

A

Once the DNA of interest has been cloned, transformed cells carrying it must be identified

26
Q

what are the selective conditions that hosts are grown in

A
  • antibiotics
  • nutrient requirements
  • plaque type
  • blue-white selection (β-galactosidase)
  • specific (hybridization, antibodies, PCR)
27
Q

why are hosts grown in selective conditions

A

allow the identification of positive clones containing the carrier vector with the DNA of interest

28
Q

what is a selectable marker

A

a gene introduced into a cell that confers a trait for artificial selection

29
Q

what is phenotypic screening

A

protein encoded by the gene changes the colour of the colony

30
Q

what occurs during blue-white screening

A
  1. lacZ gene – encodes for β-galactosidase
  2. X-Gal – substrate for the enzyme [X-gal is white but if -galactosidase is present, substrate blue]
  3. IPTG (isopropyl-[beta]-D-thiogalactopyranoside) – inducer
  4. cloning sites within the lacZ gene
  5. disruption of gene by insertion of the foreign DNA
31
Q

what occurs in the DNA ligase mechanism

A 
Energy needed for adenylation of DNA ligase reaction
ATP loses a Pi
AMP 
Release a molecule of water
Joining of DNA segments
32
Q

what are the blue colonies like in the blue-white screening

A

blue colonies have a functional β-galactosidase protein

33
Q

what are the white colonies like in blue-white screening

A

white coloines have a non-functional β-galactosidase protein

34
Q

in antibiotic resistance what plasmid does bacteria need to be resistant to Kanamycin

A

Kan ^r

35
Q

how do you read a plasmid

A

like a clock

36
Q

what is the bla gene for in a vector

A

resistance to plasmid

37
Q

what is the lacZ for in vectors

A

gives blue colour

38
Q

what is the rep gene for in vector

A

helps plasmid replicate

39
Q

what is the MCS polylinker

A

multiple cloning site

40
Q

what happens in blue-white screening when an insertion occurs

A

will get a white colour as no functional beta galactosidase protein due to insertion

41
Q

how does large scale propagation work

A

once colonies are identified, they are cultured in broth to increase numbers and therefore the amount of DNA

42
Q

how are colonies stored

A

samples are also prepared for storage at -80 degrees C

they can be kept for many years

43
Q

how is a segment of DNA cloned

A

bacterial plasmid with ampicillin resistance, lacZ gene and restriction site
isolate plasmid (vector) DNA and human DNA
insert human DNA into plasmids
add DNA ligase to bond covalently
put plasmids into lacZ bacteria by transformation
clone cells
identify clone carrying gene of interest

44
Q

what must happen to the DNA used in DNA cloning

A

cut both DNAs with same restriction enzyme so have sticky ends
mix DNAs, join by base pairing

45
Q

how are cells cloned in DNA cloning

A

plate cells onto medium with ampicillin and X-gal, identify clones of cells containing recombinant plasmids by their ability to grow in presence of ampicillin and their white colour

46
Q

why would we want to be able to clone

A

isolate DNA
establish collections
for further molecular studies

47
Q

why do we need to be able to isolate DNA

A

can then be used for making probes, to be digested, to be mapped and/or to be sequenced

48
Q

what could further molecular studies be

A

Production of special proteins
Investigation of protein/enzyme/RNA functions
Identification of mutations e.g. gene defects related to specific diseases

49
Q

what are the methods to clone DNA

A
  • restriction enzyme digestion and ligation
  • T-A cloning
  • TOPO attached unidirectional cloning
  • recombination based cloning
50
Q

what does DNA cloning require

A

segment of DNA, a host, a vector, a means to insert DNA into the vector and the vector into the host, and a selection method for the segment of interest

51
Q

why is DNA cloning useful

A

fundamental for recombinant DNA technology

Applied Genetics (20 decks)

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