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BIO107 - Cell and Molecular Biology > Finals - Gene Manipulation > Flashcards

Finals - Gene Manipulation Flashcards

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1
Q

why manipulate genes?

A
  1. understand their function and interaction with other molecules
  2. produce drugs, vaccines, hormones, and other important gene products
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2
Q

What does gene manipulation involve?

A
  1. nucleic acid hybridization
  2. rapid sequencing of all the nucleotides in a purified DNA fragment
  3. cleavage of DNA at specific sites by restriction endonucleases
  4. DNA cloning
  5. DNA engineering
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3
Q

use of probes to find a specific sequence of DNA or RNA with great accuracy and sensitivity on the basis of its ability to bind a complementary nucleic acid sequenc

A

nucleic acid hybridization

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4
Q

determine the boundaries of a gene and the amino acid sequence it encodes

A

rapid sequencing of all the nucleotides in a purified DNA fragment

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5
Q

greatly facilitates the isolation and manipulation of individual genes

A

cleavage of DNA at specific sites by restriction endonucleases

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6
Q

a single DNA molecule can be copied to generate many billios of identical molecules

A

DNA cloning

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7
Q

basic steps in DNA cloning

A
  1. isolation of DNA
  2. cleavage of DNA at particular sequences
  3. ligation of insert DNA to vector DNA
  4. introduction of recombinant DNA into compatible host cells
  5. replication and in some cases expression of recombinant DNA within host cells
  6. identification of host cells that contain recombinant DNA within host cells
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8
Q

bacteria usually used

A

E. coli

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9
Q

library of desired genes

A

genomic and cDNA library

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10
Q
  • DNA sequences are altered to make modified versions of genes, which are reinserted back into cells or organisms
  • genomic and cDNA library construction
  • methods of screening libraries
A

DNA engineering

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11
Q

methods of screening libraries

A
  1. colony and plaque hybridization
  2. chromosome walking
  3. DNA footprinting
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12
Q
  • molecular scissors that cut DNA into fragments at specific sites in their sequence
  • degrades foreign DNA -> defense vs phages
  • restriction endonucleases
A

restriction enzymes

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13
Q

restriction enzymes are aka _

A

restriction endonucleases

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14
Q

nomenclature = EcoRI

A
  • Eco = 1st letter of genus + 1st and 2nd letter of species
  • R = strain of host bacteria
  • I - order of discovery
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15
Q

Different classification of restriction enzymes

A
  1. Type I
  2. Type II
  3. Type III
  4. Type IV
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16
Q

cut DNA at random far from their recognition sequences

A

Type I

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17
Q

cut DNA at defiend positions close to or within their recognition sequences

A

Type II

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18
Q

cut DNA outside of two recognition sequences in opposite orientations

A

Type III

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19
Q

cut modified DNA (e.g. methylated)

A

Type IV

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20
Q

mechanism of restriction enzymes

A
  1. scan
  2. bind
  3. cut
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21
Q

look for a particular pattern of nucleotides

A

scan

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22
Q

responsible for scanning

A

enzyme’s recognition sequence

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23
Q

what happens after scanning?

A

enzyme will bind the DNA molecule

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24
Q

where does the restriction enzyme cut

A

in each of the two sugar-phosphate backbones of double helix

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25
Q
  • the DNA sequence to which a restriction enzyme binds
  • may be made up of 4 bases, 6 bases, or 8 bases
  • some have similar recognition sequences but different cutting sites
A

recognition sequence

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26
Q

most common no. of bases of a recognition sequence

A

6 bases

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27
Q

rare no. of bases of a recognition sequence

A

8 bases (rare cutters)

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28
Q

have similar recognition sequences but different cutting sites

A

isoschizomers

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29
Q

eg. of isoschizomers

A
  • SmaI
  • XmaI
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30
Q

restriction enzyme that produce blunt ends

A
  • AluI
  • HaeIII
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31
Q

restriction enzyme that produce sticky ends

A
  • BamHI
  • HindIII
  • EcoRI
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32
Q

cleavage is asymmetrical

A

over-hanging ends are sticky (complementary)

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33
Q

cleavage is symmetrical

A

ends are blunt

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34
Q
  • linear drawing that represents the location of all RE recognition sites within a piece of DNA
  • usually the first step in characterizing an unknown DNA, and a prerequisite to manipulating it for other purposes
A

restriction mapping

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35
Q

Two ways of restriction mapping

A
  1. DNA sequence is known
  2. DNA sequence is unknown
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36
Q
  • search for specific enzyme recognition site
  • draw map based on distances
A

DNA sequence in known

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37
Q
  • compare pattern of DNA fragments
  • single digests show how many sites are present
  • double digests show where the sites are relative to one another
A

DNA sequence is unknown

38
Q

show how many sites are present

A

single digests

39
Q

show where the sites are relative to one another

A

double digests (cutting with 2 enzymes)

40
Q
  • made by fusing a DNA-binding domain to a nuclease domain
  • can target large DNA sites
  • can be engineered to bind desired DNA sequences
A

artificial restriction enzymes

41
Q

how are artificial restriction enzymes made

A

fuse a DNA-binding domain to a nuclease domain

42
Q

artificial restriction enzymes can target large DNA sites up to __ bp

A

36

43
Q

e.g. of artificial restriction enzymes

A
  1. zinc finger nucleases
  2. TALENs
  3. CRISPR
44
Q
  • joins broken phosphodiester linkages in the sugar-phosphate backbone
  • common source: T4 bacteriophage
  • can ligate sticky-ends more efficiently than blunt-ends
A

DNA ligase

45
Q

common source of DNA ligase

A

T4 bacteriophage

46
Q

addition of poly(dA) tail or poly (dT) tail to ends of the DNA from each source

A

modified blunt-end ligation

47
Q

DNA molecules in which foreign DNA molecule is inserted and which is further capable of replication within host cell to produce multiple clones of the recombinant DNA

A

cloning vector

48
Q

Main features of cloning vectors

A
  1. origin of replication
  2. multiple cloning site
  3. selectable marker(s)
49
Q
  • DNA segment with several RE sites located next to each other
  • also called as polylingker
A

multiple cloning site

50
Q

multiple cloning site is also called as

A

polylinker

51
Q

REs are __ __ anywhere else in the vector plasmid

A

not present

52
Q

cutting the vector with and RE that recognize a site in the polylinker…

A

does not disrupt any of essential features of the vector

53
Q
  • allows selection of host cells with the recombinant vector
  • basis if recombinant DNA is formed
A

selectable marker(s)

54
Q
  • uses selectable marker lacZ
  • if ligation and transformation is successful, bacterial colony will be white
  • if not, colony will be blue
  • transformed cells are grown in the presence of X-gal
A

blue-white screening

55
Q

selectable marker of blue-white screening

A

lacZ

56
Q

color if ligation and transformation is successful

A

white

57
Q

color if ligation and transformation is unsuccessful

A

blue

58
Q

lactose/galactose analog

A

IPTG

59
Q

in blue-white screening, transformed cells are grown in the presence of __

A

X-gal

60
Q

white colony

A

5-bromo-4-chloro-indoxyl

61
Q

blue colony

A

5,5’-dibromo-4,4’-dichloro-indigo

62
Q

gene in lacZ

A

beta-galactosidase

63
Q

types of cloning vectors

A
  1. plasmids
  2. bacteriophage lambda
64
Q
  • naturally occuring extrachromosomal circular ds DNA molecules that carry an origin of replication and replicate autonomously within bacterial cells
  • pBR322
A

plasmids

65
Q

plasmid and was one of the first widely used E. coli cloning vectors

A

pBR322

66
Q

what does pBR322 contain

A
  1. ampicillin-resistance gene
  2. tetracycline-resistance gene
  3. Col E1 replication origin
  4. Eco RI site
67
Q
  • linear, double-stranded molecule
  • with cos site
  • particularly useful for preparing genomic libraries, because they can hold a larger piece of DNA than a plasmid vector
A

bacteriophage lambda

68
Q
  • single-stranded
  • complementary ends/cohesive ends
  • can hybridize with each other
A

cos site in bacteriophage lambda

69
Q

other types of cloning vectors

A
  1. cosmid
  2. BAC (bacterial artificial chromosome)
  3. YAC (yeast artificial chromosome)
  4. MAC (mammalian artificial chromosome)
70
Q

size limits of insert of plasmid

A

less than or equal to 10 kb

71
Q

size limits of insert of phage

A

5-20 kb

72
Q
  • plasmid containing a bacteriophage λ cos site
  • 35-45 kb
  • genomic library construction
A

cosmid

73
Q
  • E.coli F factor plasmid
  • 75-300kb
  • analysis of large genomes
A

BAC (bacterial artificial chromosome)

74
Q
  • yeast centromere, telomere, and autonomously replicating sequence
  • 100-1000 kb (1Mb)
  • analysis of large genomes, YAC transgenic mice
A

YAC (yeast artificial chromosome)

75
Q
  • mammalian centromere, telomere, and origin of replication
  • 100kb to > 1Mb
  • under development for use in animal biotechnology and human gene therapy
A

MAC (mammalian artificial chromosome)

76
Q

size limits of insert of cosmoid

A

35-45 kb

77
Q

size limits of insert of BAC

A

75-300 kb

78
Q

size limits of insert of YAC

A

100kb - 1Mb

79
Q

size limits of insert of MAC

A

100 kb to > 1Mb

80
Q
  • cloning vector with regulatory regions for gene expression
  • selection is for cells expressing the gene
A

expression vectors

81
Q

what are expression vectors

A

cloning vectors with regulatory regions for gene expression (e.g. strong promoter)

82
Q

Methods of introducing recombinant DNA to target host cells

A
  1. tranformation
  2. infection of bacterial cells by bacteriophage
  3. microinjection
  4. electroporation
  5. gene gun or bioballistic method
83
Q
  • transfer of cell-free or exogenous DNA into bacterial host cell
  • prereq: cells must be competent
A

transformation

84
Q

prereq of transformation

A

cell must be competent
- CaCl2

85
Q
  • uses short electrical high volatege pulses -> transient pores
  • the DNA then enters the cell
A

electroporation

86
Q

what is produced during the short electrical high voltage pulses

A

transient pores

87
Q

parts of a bacteriophage

A
  1. protein coat
  2. DNA
  3. sheath
  4. core
88
Q
  • requires the use of a glass micropipette with a diameter that is much smaller than the cell
  • the micropipette punctures the plasma membrane, and DNA can be injected through it
A

microinjection

89
Q

foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles and bombarded onto the target tissue or cells using a particle gun

A

gene gun

90
Q

where is DNA placed during gene gunning

A

coated onto surface of gold or tungsten particles

91
Q

size of gold or tungsten particles used

A

1-3 micrometers

BIO107 - Cell and Molecular Biology (21 decks)

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