Finals - Gene Manipulation Flashcards
1
Q
why manipulate genes?
A
- understand their function and interaction with other molecules
- produce drugs, vaccines, hormones, and other important gene products
2
Q
What does gene manipulation involve?
A
- nucleic acid hybridization
- rapid sequencing of all the nucleotides in a purified DNA fragment
- cleavage of DNA at specific sites by restriction endonucleases
- DNA cloning
- DNA engineering
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
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
5
Q
greatly facilitates the isolation and manipulation of individual genes
A
cleavage of DNA at specific sites by restriction endonucleases
6
Q
a single DNA molecule can be copied to generate many billios of identical molecules
A
DNA cloning
7
Q
basic steps in DNA cloning
A
- isolation of DNA
- cleavage of DNA at particular sequences
- ligation of insert DNA to vector DNA
- introduction of recombinant DNA into compatible host cells
- replication and in some cases expression of recombinant DNA within host cells
- identification of host cells that contain recombinant DNA within host cells
8
Q
bacteria usually used
A
E. coli
9
Q
library of desired genes
A
genomic and cDNA library
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
11
Q
methods of screening libraries
A
- colony and plaque hybridization
- chromosome walking
- DNA footprinting
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
13
Q
restriction enzymes are aka _
A
restriction endonucleases
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
15
Q
Different classification of restriction enzymes
A
- Type I
- Type II
- Type III
- Type IV
16
Q
cut DNA at random far from their recognition sequences
A
Type I
17
Q
cut DNA at defiend positions close to or within their recognition sequences
A
Type II
18
Q
cut DNA outside of two recognition sequences in opposite orientations
A
Type III
19
Q
cut modified DNA (e.g. methylated)
A
Type IV
20
Q
mechanism of restriction enzymes
A
- scan
- bind
- cut
21
Q
look for a particular pattern of nucleotides
A
scan
22
Q
responsible for scanning
A
enzyme’s recognition sequence
23
Q
what happens after scanning?
A
enzyme will bind the DNA molecule
24
Q
where does the restriction enzyme cut
A
in each of the two sugar-phosphate backbones of double helix
25
- 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
recognition sequence
26
most common no. of bases of a recognition sequence
6 bases
27
rare no. of bases of a recognition sequence
8 bases (rare cutters)
28
have similar recognition sequences but different cutting sites
isoschizomers
29
eg. of isoschizomers
- SmaI
- XmaI
30
restriction enzyme that produce blunt ends
- AluI
- HaeIII
31
restriction enzyme that produce sticky ends
- BamHI
- HindIII
- EcoRI
32
cleavage is asymmetrical
over-hanging ends are sticky (complementary)
33
cleavage is symmetrical
ends are blunt
34
- 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
restriction mapping
35
Two ways of restriction mapping
1. DNA sequence is known
2. DNA sequence is unknown
36
- search for specific enzyme recognition site
- draw map based on distances
DNA sequence in known
37
- compare pattern of DNA fragments
- single digests show how many sites are present
- double digests show where the sites are relative to one another
DNA sequence is unknown
38
show how many sites are present
single digests
39
show where the sites are relative to one another
double digests (cutting with 2 enzymes)
40
- made by fusing a DNA-binding domain to a nuclease domain
- can target large DNA sites
- can be engineered to bind desired DNA sequences
artificial restriction enzymes
41
how are artificial restriction enzymes made
fuse a _DNA-binding_ _domain_ to a _nuclease_ _domain_
42
artificial restriction enzymes can target large DNA sites up to __ bp
36
43
e.g. of artificial restriction enzymes
1. zinc finger nucleases
2. TALENs
3. CRISPR
44
- joins broken phosphodiester linkages in the sugar-phosphate backbone
- common source: T4 bacteriophage
- can ligate sticky-ends more efficiently than blunt-ends
DNA ligase
45
common source of DNA ligase
T4 bacteriophage
46
addition of poly(dA) tail or poly (dT) tail to ends of the DNA from each source
modified blunt-end ligation
47
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
cloning vector
48
Main features of cloning vectors
1. origin of replication
2. multiple cloning site
3. selectable marker(s)
49
- DNA segment with several RE sites located next to each other
- also called as polylingker
multiple cloning site
50
multiple cloning site is also called as
polylinker
51
REs are __ __ anywhere else in the vector plasmid
not present
52
cutting the vector with and RE that recognize a site in the polylinker...
does not disrupt any of essential features of the vector
53
- allows selection of host cells with the recombinant vector
- basis if recombinant DNA is formed
selectable marker(s)
54
- 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
blue-white screening
55
selectable marker of blue-white screening
lacZ
56
color if ligation and transformation is successful
white
57
color if ligation and transformation is unsuccessful
blue
58
lactose/galactose analog
IPTG
59
in blue-white screening, transformed cells are grown in the presence of __
X-gal
60
white colony
5-bromo-4-chloro-indoxyl
61
blue colony
5,5'-dibromo-4,4'-dichloro-indigo
62
gene in lacZ
beta-galactosidase
63
types of cloning vectors
1. plasmids
2. bacteriophage lambda
64
- naturally occuring extrachromosomal circular ds DNA molecules that carry an origin of replication and replicate autonomously within bacterial cells
- pBR322
plasmids
65
plasmid and was one of the first widely used E. coli cloning vectors
pBR322
66
what does pBR322 contain
1. ampicillin-resistance gene
2. tetracycline-resistance gene
3. Col E1 replication origin
4. Eco RI site
67
- 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
bacteriophage lambda
68
- single-stranded
- complementary ends/cohesive ends
- can hybridize with each other
cos site in bacteriophage lambda
69
other types of cloning vectors
1. cosmid
2. BAC (bacterial artificial chromosome)
3. YAC (yeast artificial chromosome)
4. MAC (mammalian artificial chromosome)
70
size limits of insert of plasmid
less than or equal to 10 kb
71
size limits of insert of phage
5-20 kb
72
- plasmid containing a bacteriophage λ cos site
- 35-45 kb
- genomic library construction
cosmid
73
- E.coli F factor plasmid
- 75-300kb
- analysis of large genomes
BAC (bacterial artificial chromosome)
74
- yeast centromere, telomere, and autonomously replicating sequence
- 100-1000 kb (1Mb)
- analysis of large genomes, YAC transgenic mice
YAC (yeast artificial chromosome)
75
- mammalian centromere, telomere, and origin of replication
- 100kb to > 1Mb
- under development for use in animal biotechnology and human gene therapy
MAC (mammalian artificial chromosome)
76
size limits of insert of cosmoid
35-45 kb
77
size limits of insert of BAC
75-300 kb
78
size limits of insert of YAC
100kb - 1Mb
79
size limits of insert of MAC
100 kb to > 1Mb
80
- cloning vector with regulatory regions for gene expression
- selection is for cells expressing the gene
expression vectors
81
what are expression vectors
cloning vectors with regulatory regions for gene expression (e.g. strong promoter)
82
Methods of introducing recombinant DNA to target host cells
1. tranformation
2. infection of bacterial cells by bacteriophage
3. microinjection
4. electroporation
5. gene gun or bioballistic method
83
- transfer of cell-free or exogenous DNA into bacterial host cell
- prereq: cells must be competent
transformation
84
prereq of transformation
cell must be competent
- CaCl2
85
- uses short electrical high volatege pulses -> transient pores
- the DNA then enters the cell
electroporation
86
what is produced during the short electrical high voltage pulses
transient pores
87
parts of a bacteriophage
1. protein coat
2. DNA
3. sheath
4. core
88
- 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
microinjection
89
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
gene gun
90
where is DNA placed during gene gunning
coated onto surface of gold or tungsten particles
91
size of gold or tungsten particles used
1-3 micrometers
