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Protein and Nucleic acid > Recombinant DNA > Flashcards

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

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2
Q

Blank can be constructed
by ligating different fragments
together

A

artificial plasmids

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2
Q

plasmids that are able to perpetuate themselves in Blank, are the Blank of recombinant DNA
technology

A

E. coli, workhorses

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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

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4
Q

3 common features of plasmids useful
as cloning vectors:

A
  • replicator
  • selectable marker
  • cloning site
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5
Q
  • an origin of replication, or ori
    selectable marker
  • a gene conferring resistance to an
    antibiotic
A

replicator

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6
Q

only cells containing the Blank will grow in the presence of the antibiotic

A

cloning vector

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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

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8
Q
  • hybrid DNA molecules consisting of plasmid DNA sequences plus
    inserted DNA elements (inserts)
  • chimeric constructs or chimeric plasmids
A

recombinant plasmids

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9
Q

TRUE OR FALSE
presence of foreign DNA sequences does affect replication of the plasmid

A

False (not adversely)

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10
Q

virtually any DNA sequence can be
Blank and Blank

A

selectively cloned, amplified

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11
Q

DNA sequences that are difficult to
clone

A
  • inverted repeats
  • origins of replication
  • centromeres
  • telomeres
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12
Q

TRUE OR FALSE
most plasmids with inserts > 10
kbp are replicated efficiently

A

False (not replicated)

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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

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14
Q
  • an alternative method for joining
    different DNAs
A

blunt-end ligation

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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

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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

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17
Q
  • a short region of DNA sequence
    bearing numerous restriction sites
A

polylinker cloning site

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17
Q

cleavage of the ligated DNA with the
restriction enzyme leaves Blank sticky ends useful in cloning reactions

A

tailor-made

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18
Q
  • sometimes it is desirable to insert
    the DNA in a particular orientation
A

Promoters and Directional Cloning

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19
Q

TRUE OR FALSE
the DNA must be placed
upstream from a promoter

A

False (downstream)

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20
Q
  • a nucleotide sequence lying upstream of a gene
  • controls expression of the gene
A

promoter

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20
Q
  • bind specifically at promoters and
    initiate transcription of adjacent
    genes, copying template DNA into
    RNA products
A

RNA polymerase molecules

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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

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22
Q

TRUE OR FALSE
ligation of such molecules into the
plasmid vector can only take place in two orientation to give directional cloning

A

False (one)

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23
Q

TRUE OR FALSE
the foreign DNA and the plasmid are digested with the same 3 enzymes

A

False (2)

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24
Q

pUC

A

(universal cloning plasmid)

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24
Q

the first biologically functional
chimeric DNA molecules constructed
in vitro were assembled from parts
of different plasmids in 1973 by ?

A

Stanley Cohen, Annie Chang, Herbert
Boyer, and Robert Helling

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25
Q
  • uptake and replication of exogenous
    DNA by a recipient cell
A

transformation

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26
Q

to facilitate transformation, the
bacterial cells were rendered somewhat permeable to DNA by Blank and a brief Blank

A

Ca2+ treatment, 42°C heat
shock

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27
Q
  • 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)
A

shuttle vectors

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27
Q

eukaryotic genes can be cloned in ?

A

bacterial hosts

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28
Q

DNA molecules 2 megabase pairs in
length have been successfully
propagated in yeast by creating ?

A

yeast artificial chromosomes (YACs)

29
Q
  • provide the site for attachment of the chromosome to the spindle during mitosis and meiosis
A

centromeres

30
Q
  • nucleotide sequences defining the ends of chromosomes
  • essential for proper replication of the chromosome
A

telomeres

31
Q
  • a set of cloned fragments that
    collectively represent the genes of a
    specific organism
A

DNA library

31
Q
  • offer the advantage of introducing large amounts of recombinant DNA into human cells, ideally for the
    treatment of disease
A

HACs

32
Q

carry Blank of DNA, centromeres, telomeres, and replication origins

A

5 to 10 x 103 kbp (normal human chromosomes have 50 to 250 x 103 kbp of DNA)

33
Q
  • clone all the genes of the organism at one time
  • at least onerecombinant clone will contain at least part of the gene of
    interest
A

shotgun cloning

34
Q
  • a common method of screening genomic libraries
A

colony hybridization experiment

35
Q
  • a single-stranded DNA of defined sequence that is distinctively labeled
    with some easily detectable marker,
    such as a fluorescent tag
A

probe

36
Q
  • a technique for identifying and
    dramatically amplifying the amount
    of a specific DNA segment
A

polymerase chain reaction (PCR)

37
Q

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

A

template for DNA polymerase, primers

38
Q

the use of heat-stable DNA polymerases from Blank makes it unnecessary to add fresh enzyme for each round of synthesis

A

thermophilic bacteria (Taq DNA polymerase from Thermus aquaticus)

39
Q

25 rounds would increase its concentration about Blank times

A

33 million

40
Q

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

A

False ( can be analyzed )

41
Q
  • DNA molecules copied from mRNA
    templates
A

complementary DNAs (cDNAs)

42
Q

TRUE OR FALSE
most eukaryotic mRNAs carry 5’- poly(A) tails

A

False (3’- poly)

43
Q
  • constructed by synthesizing cDNA
    from purified cellular mRNA
  • an alternative strategy for gene
    isolation, especially eukaryotic gene
A

cDNA libraries

44
Q
  • an enzyme that synthesizes a DNA
    strand, copying RNA as the template
A

reverse transcriptase

45
Q
  • a variation on the basic PCR method
  • useful for cloning the gene that
    encodes a specific mRNA
A

RT-PCR (reverse transcriptase-PCR)

46
Q

this amplified DNA is then used to probe Blank to isolate the gene of interest

A

genomic libraries

46
Q
  • used to synthesize a cDNA strand
    complementary with the RNA, and
    this cDNA serves as the template for
    further cycles of PCR
A

Reverse transcriptase (RT)

47
Q
  • 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
A

Expressed Sequence Tags (EST)

48
Q

Blank are Arrays of DifferentOligonucleotides
Immobilized on a Chip

A

DNA Microarrays (Gene Chips)

49
Q

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

A

robotic, 2-dimensional

50
Q

TRUE OR FALSE
synthesis is performed by
photochemical chemistry adapted into a phosphoramidite process that can be controlled by light

A

False (phosphoramidite , photochemical)

51
Q

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

A

gene chips

52
Q

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

A

25 nucleotides,

53
Q

the Blank on such gene chips are used as the probes in a hybridization experiment to reveal gene expression patterns

A

oligonucleotides

54
Q

Introducing changes into Expressed Proteins

A

In Vitro Mutagenesis

55
Q
  • mutations introduced into the gene
  • alter the nucleotide sequence of a
    cloned gene systematically
A

in vitro mutagenesis

56
Q
  • mutant primers are added to a PCR
    rxn in which the gene (or segment of
    a gene) is undergoing amplification
A

PCR-based mutagenesis

57
Q
  • 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
A

mutant primers

58
Q

are widely used as
experimental animals to investigate
models of human disease and
physiology

A

transgenic mice

59
Q

Blank and Blank to correct
certain human genetic disorders

A

gene replacement therapy (gene
therapy), gene editing

60
Q
  • repair the damage caused by a
    genetic deficiency through intro of a
    functional version of the defective
    gene
A

Human gene therapy

61
Q

the nucleotide sequence of an
Blank or Blank is edited to change it into one
with normal function

A

inactive, dysfunctional resident
gene

62
Q

treat cancer through expression of
the Blank and Blank tumor suppressor
genes

A

E1A, and p53

63
Q

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

A

Kymriah therapy

64
Q

as Vectors in Human Gene
Therapy

A

Viruses

65
Q
  • introduce a vector carrying the
    expression cassette into cells that
    have been isolated from a patient
    and cultured in the laboratory
A

ex vivo route

66
Q
  • commonly used in gene therapy
A

retroviruses

67
Q

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

A

False (expression cassette, cDNA version)

68
Q

TRUE OR FALSE
sickle-cell anemia is due to a single AA
change in the primary structure of the
A-globin subunit of Hb

A

False (β-globin)

69
Q

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

A

False (T, A)

70
Q

TRUE OR FALSE
this change transforms the 7th codon
nucleotide sequence from GAG,
which codes for Glu, to GTG, which
codes for Val

A

False (6th codon)

71
Q

is a system that can be
used to carry out such genome
editing
- can target and edit any
nucleotide sequence in any genome

A

CRISPR-Cas9

72
Q
  • clustered regularly interspaced short
    palindromic repeats
  • a family of DNA repeats that are
    widespread among prokaryotic
    genomes
A

CRISPR

73
Q
  • 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
A

Cas9 (CRISPR-associated protein 9)

74
Q

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

A

complementary base-pairing

Protein and Nucleic acid (6 decks)

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