Recombinant DNA Flashcards
1
Q
naturally occurring, circular,
extrachromosomal DNA molecules.
* carry genes specifying novel
metabolic activities that are
advantageous to the host bacterium.
A
Plasmids
2
Q
Blank can be constructed
by ligating different fragments
together
A
artificial plasmids
2
Q
plasmids that are able to perpetuate themselves in Blank, are the Blank of recombinant DNA
technology
A
E. coli, workhorses
3
Q
Blank could be inserted into artificial plasmids and
carried into E. coli and propagated as
part of the plasmid
A
“foreign” DNA sequences
4
Q
3 common features of plasmids useful
as cloning vectors:
A
- replicator
- selectable marker
- cloning site
5
Q
- an origin of replication, or ori
selectable marker - a gene conferring resistance to an
antibiotic
A
replicator
6
Q
only cells containing the Blank will grow in the presence of the antibiotic
A
cloning vector
7
Q
- a sequence of nucleotides
representing one or more restriction
endonuclease cleavage sites - located where the insertion of foreign
DNA neither disrupts the plasmid’s
ability to replicate nor inactivates
essential markers
A
cloning site
8
Q
- hybrid DNA molecules consisting of plasmid DNA sequences plus
inserted DNA elements (inserts) - chimeric constructs or chimeric plasmids
A
recombinant plasmids
9
Q
TRUE OR FALSE
presence of foreign DNA sequences does affect replication of the plasmid
A
False (not adversely)
10
Q
virtually any DNA sequence can be
Blank and Blank
A
selectively cloned, amplified
11
Q
DNA sequences that are difficult to
clone
A
- inverted repeats
- origins of replication
- centromeres
- telomeres
12
Q
TRUE OR FALSE
most plasmids with inserts > 10
kbp are replicated efficiently
A
False (not replicated)
13
Q
- joining the ends of the foreign DNA
insert to the ends of a linearized
plasmid - facilitated if the ends of the plasmid
and the insert have complementary,
single-stranded overhangs - these ends can base-pair with one
another, annealing the 2 molecules
together
A
Construction of Chimeric Plasmids
14
Q
- an alternative method for joining
different DNAs
A
blunt-end ligation
15
Q
- most widely used DNA ligase
- an ATP-dependent enzyme that can
even ligate 2 DNA fragments whose
ends lack overhangs (blunt-ended
DNAs)
A
bacteriophage T4 DNA ligase
16
Q
- short synthetic DNA duplexes whose
nucleotide sequence consists of little
more than a restriction site and can be
blunt-end ligated onto any DNA
A
linkers
17
Q
- a short region of DNA sequence
bearing numerous restriction sites
A
polylinker cloning site
17
Q
cleavage of the ligated DNA with the
restriction enzyme leaves Blank sticky ends useful in cloning reactions
A
tailor-made
18
Q
- sometimes it is desirable to insert
the DNA in a particular orientation
A
Promoters and Directional Cloning
19
Q
TRUE OR FALSE
the DNA must be placed
upstream from a promoter
A
False (downstream)
20
Q
- a nucleotide sequence lying upstream of a gene
- controls expression of the gene
A
promoter
20
Q
- bind specifically at promoters and
initiate transcription of adjacent
genes, copying template DNA into
RNA products
A
RNA polymerase molecules
21
Q
- DNA molecules whose ends have different overhangs can be used
to form chimeric constructs in which the foreign DNA can enter
the plasmid in only one orientation
A
Directional cloning
22
TRUE OR FALSE
ligation of such molecules into the
plasmid vector can only take place in two orientation to give directional cloning
False (one)
23
TRUE OR FALSE
the foreign DNA and the plasmid are digested with the same 3 enzymes
False (2)
24
pUC
(universal cloning plasmid)
24
the first biologically functional
chimeric DNA molecules constructed
in vitro were assembled from parts
of different plasmids in 1973 by ?
Stanley Cohen, Annie Chang, Herbert
Boyer, and Robert Helling
25
* uptake and replication of exogenous
DNA by a recipient cell
transformation
26
to facilitate transformation, the
bacterial cells were rendered somewhat permeable to DNA by Blank and a brief Blank
Ca2+ treatment, 42°C heat
shock
27
* plasmids capable of propagating and
transferring (“shuttling”)genes
between 2 different organisms, one
of which is typically a prokaryote (E.
coli) and the other a eukaryote
(yeast)
shuttle vectors
27
eukaryotic genes can be cloned in ?
bacterial hosts
28
DNA molecules 2 megabase pairs in
length have been successfully
propagated in yeast by creating ?
yeast artificial chromosomes (YACs)
29
* provide the site for attachment of the chromosome to the spindle during mitosis and meiosis
centromeres
30
* nucleotide sequences defining the ends of chromosomes
* essential for proper replication of the chromosome
telomeres
31
* a set of cloned fragments that
collectively represent the genes of a
specific organism
DNA library
31
* offer the advantage of introducing large amounts of recombinant DNA into human cells, ideally for the
treatment of disease
HACs
32
carry Blank of DNA, centromeres, telomeres, and replication origins
5 to 10 x 103 kbp (normal human chromosomes have 50 to 250 x 103 kbp of DNA)
33
* clone all the genes of the organism at one time
* at least onerecombinant clone will contain at least part of the gene of
interest
shotgun cloning
34
* a common method of screening genomic libraries
colony hybridization experiment
35
* a single-stranded DNA of defined sequence that is distinctively labeled
with some easily detectable marker,
such as a fluorescent tag
probe
36
* a technique for identifying and
dramatically amplifying the amount
of a specific DNA segment
polymerase chain reaction (PCR)
37
a preparation of denatured DNA
containing the desired segment (e.g.
total genomic DNA) serves as a
Blank, and
two specific oligonucleotides serve
as Blank
template for DNA polymerase, primers
38
the use of heat-stable DNA polymerases from Blank makes it unnecessary to add fresh enzyme for each round of synthesis
thermophilic bacteria (Taq DNA polymerase from Thermus aquaticus)
39
25 rounds would increase its concentration about Blank times
33 million
40
TRUE OR FALSE
with PCR techniques, DNA from a single hair or sperm cannot be analyzed to identify particular individuals in criminal cases without ambiguity
False ( can be analyzed )
41
* DNA molecules copied from mRNA
templates
complementary DNAs (cDNAs)
42
TRUE OR FALSE
most eukaryotic mRNAs carry 5'- poly(A) tails
False (3’- poly)
43
* constructed by synthesizing cDNA
from purified cellular mRNA
* an alternative strategy for gene
isolation, especially eukaryotic gene
cDNA libraries
44
* an enzyme that synthesizes a DNA
strand, copying RNA as the template
reverse transcriptase
45
* a variation on the basic PCR method
* useful for cloning the gene that
encodes a specific mRNA
RT-PCR (reverse transcriptase-PCR)
46
this amplified DNA is then used to probe Blank to isolate the gene of interest
genomic libraries
46
* used to synthesize a cDNA strand
complementary with the RNA, and
this cDNA serves as the template for
further cycles of PCR
Reverse transcriptase (RT)
47
* short (200 nucleotides) sequences obtained by determining a portion of
the nucleotide sequence for each insert in randomly selected cDNAs
* represents part of a gene that is
being expressed
Expressed Sequence Tags (EST)
48
Blank are Arrays of DifferentOligonucleotides
Immobilized on a Chip
DNA Microarrays (Gene Chips)
49
Blank methods can be used to synthesize combinatorial libraries of
DNA oligonucleotides directly on a solid support, such that the
completed library is a Blank array of different oligonucleotides
robotic, 2-dimensional
50
TRUE OR FALSE
synthesis is performed by
photochemical chemistry adapted into a phosphoramidite process that can be controlled by light
False (phosphoramidite , photochemical)
51
the final products of such procedures are referred to as Blank because the oligonucleotide sequences synthesized upon the chip
represent the sequences of chosen genes
gene chips
52
the oligonucleotides are up to Blank long and as many as 1.3 million different oligonucleotides
can be arrayed on a chip 1 cm square
25 nucleotides,
53
the Blank on such gene chips are used as the probes in a hybridization experiment to reveal gene expression patterns
oligonucleotides
54
Introducing changes into Expressed Proteins
In Vitro Mutagenesis
55
* mutations introduced into the gene
* alter the nucleotide sequence of a
cloned gene systematically
in vitro mutagenesis
56
* mutant primers are added to a PCR
rxn in which the gene (or segment of
a gene) is undergoing amplification
PCR-based mutagenesis
57
* primers whose sequence has been
specifically altered to introduce a
directed change at a particular place
in the nucleotide sequence of the
gene being amplified
mutant primers
58
are widely used as
experimental animals to investigate
models of human disease and
physiology
transgenic mice
59
Blank and Blank to correct
certain human genetic disorders
gene replacement therapy (gene
therapy), gene editing
60
* repair the damage caused by a
genetic deficiency through intro of a
functional version of the defective
gene
Human gene therapy
61
the nucleotide sequence of an
Blank or Blank is edited to change it into one
with normal function
inactive, dysfunctional resident
gene
62
treat cancer through expression of
the Blank and Blank tumor suppressor
genes
E1A, and p53
63
insertion of the CD19 surface antigen
gene into a patient’s T lymphocytes
has been used to create the Blank for treatment of lymphoma and leukemia
Kymriah therapy
64
as Vectors in Human Gene
Therapy
Viruses
65
* introduce a vector carrying the
expression cassette into cells that
have been isolated from a patient
and cultured in the laboratory
ex vivo route
66
* commonly used in gene therapy
retroviruses
67
TRUE OR FALSE
* the gene is typically in the form of an
cDNA version consisting of a
expression cassette of the gene
downstream from a promoter that
will drive expression of the gene
False (expression cassette, cDNA version)
68
TRUE OR FALSE
sickle-cell anemia is due to a single AA
change in the primary structure of the
A-globin subunit of Hb
False (β-globin)
69
TRUE OR FALSE
this mutation occurs when a A is
substituted for T as the middle base in
the β-globin gene’s 6th codon
False (T, A)
70
TRUE OR FALSE
this change transforms the 7th codon
nucleotide sequence from GAG,
which codes for Glu, to GTG, which
codes for Val
False (6th codon)
71
is a system that can be
used to carry out such genome
editing
- can target and edit any
nucleotide sequence in any genome
CRISPR-Cas9
72
* clustered regularly interspaced short
palindromic repeats
* a family of DNA repeats that are
widespread among prokaryotic
genomes
CRISPR
73
* an RNA-guided endonuclease
* an RNA-binding enzyme that
catalyzes the cleavage of dsDNA at a
specific site determined by the
nucleotide sequence of its bound
RNA
Cas9 (CRISPR-associated protein 9)
74
through Blank, this guide RNA (gRNA, sgRNA
for single guide RNA) can deliver the
Cas9 endonuclease to any desired
nucleotide sequence within a target
DNA
complementary base-pairing
