Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Biotechnology I > Ch. 4 Synthesis In Vivo And In Vitro > Flashcards

Ch. 4 Synthesis In Vivo And In Vitro Flashcards

1
Q

Which of the following enzymes aid in uncoiling DNA?

A 
All of the above
DNA gyrase
DNA helicase
Topoisomerase IV
Single-stranded binding protein
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Why is an RNA primer necessary during replication?

A

DNA polymerase III requires a 3’-OH to elongate DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the functions of the two essential subunits of DNA polymerase III?

A

One subunit links nucleotides and the other ensures accuracy

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Which of the following statements about mismatch repair is incorrect?

A

MutSHL can synthesize new DNA after a mismatch has been excised

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Which of the following statements is incorrect regarding DNA replication?

A

Prokaryotic chromosomes have multiple origins of replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

During in vitro DNA replication, which of the following components is not required?

A

DNA helicase to separate the strands

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Which of the following is not a step in the chemical synthesis of DNA?

A

The 3’ phosphate group is added using phosphorylase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

During chemical synthesis of DNA, a portion of the nucleotides does not react. How can the efficiency of such reactions be increased?

A

The desired oligonucleotide can be separated from the trucated oligos by electrophoresis.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Which of the following components terminates the chain in a sequencing reaction?

A

Dideoxynucleotides

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Which of the following statements about PCR is incorrect?

A

The DNA template is denatured using helicase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Which of the following is not an advantage of automated cycle sequencing over the chain termination method of sequencing?

A

All of the above are advantages

  • the reactions in an automated sequencer can be performed faster
  • the reactions performed in a automated sequencer can be read by a computer rather than a human
  • higher temperatures are used during cycle sequencing, which prevent secondary structures from forming in the DNA and early termination of the reaction
  • in cycle sequencing, nonspecific interactions by the primer can be controlled by raising the annealing temperature
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Which of the following statements about degenerate primers is not correct?

A

Degenerate primers are used even when the sequence of DNA is known

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Which of the following techniques would allow a researcher to determine the genetic relatedness between two samples of DNA?

A

Randomly amplified polymorphic DNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Why would a researcher want to use RT-PCR?

A

RT-PCR generates a DNA molecule without the noncoding introns from eukaryotic mRNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Which of the following is an application for PCR?

A

All of the above

  • Site-directed mutagenesis
  • creation of insertions, deletions, and fusions of different gene segments
  • amplification of specific segments of DNA
  • for cloning into vectors
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

In ___ sequencing, the DNA fragments are found to a solid surface via a flow cell

A

Illumina

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Flashes of light are emitted whenever a base is added in ___ sequencing.

A

454

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Replication starts at an ___ __ ___.

A

Origin of replication (ori)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Replication of DNA

DNA polymerase synthesizes the ___ ___ as one continuous piece.

DNA polymerase synthesizes the ___ ___ as Okazaki fragments.

A

Leading strand; lagging strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Semiconservative replication -

A

Each copy has one strand from the original helix and one new strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Replication of DNA

DNA ___ removes the supercoiling.

___ unwinds the double helix.

A

Gyrase; helicase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Replication of DNA

___-___ ___ ___ coats the single-stranded regions.

A

Single-stranded binding protein

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Replication of DNA

DNA ___ links the 3’-OH and 5’-PO4 of neighboring nucleotides, forming ___ ___.

A

Ligase; phosphodiester bond

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Replication of DNA

The final step is to add ___ ___ along the new strand.

A

Methyl groups

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

Replication of DNA

There is a delay in methylating the new strand, thus, the DNA double helix is ___.

A

Hemimethylated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Replication of DNA

___ ___ and ___ ___ add the methyl groups onto the newly synthesized DNA strand.

A

DAM methylase and DCM methylase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

Replication of DNA

The two daughter copies of circular chromosomes may become ___, or connected like two links of chain.

A

Catenated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

Replication of DNA

___ __ untangles the two chromosomes so they can partition into the daughter cells.

A

Topoisomerase IV

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

Replication of DNA

DNA polymerase cannot synthesize new DNA without a pre-existing ___.

A

3’-OH

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

Replication of DNA

DNA replication requires an ___ ___ to initiate strand formation.

A

RNA primer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

Replication of DNA

The ___ protein displaces the ___ proteins.

A

PriA; SSB

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

Replication of DNA

___ associates with the PriA protein.

A

Primase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

Replication of DNA

The ___ makes the short DNA primer.

A

Primase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

Replication of DNA

The ___ ___ is composed of alternating Okazaki fragments and RNA primers.

A

Lagging strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

Replication of DNA

___ ___ binds to the primer region, and as it moves forward, it degrades the RNA and replaces it with DNA.

A

DNA polymerase I

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

Replication of DNA

___ ___ seal the nick in the phosphate backbone.

A

DNA ligase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
37
Q

Replication of DNA

Unwinds in one direction, synthesizes in opposite direction

A

Lagging strand

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
38
Q

Replication of DNA

In E. coli, ___ ___ ___ identify a mistake in replication, excise the new nucleotides around the mistake, and recruit ___ ___ to a single-stranded region to make the new strand without a mistake.

A

Mismatch repair proteins (MutSHL); DNA polymerase III

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
39
Q

Replication of DNA

___ ___ recognizes the bulge or distortion in the sequence.

___ finds the nearest GATC site and nicks the non-methylated strand.

___ holds them together.

___ ___ corrects the sequence.

A

MutS protein

MutH

MutL

DNA polymerase III

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
40
Q

Replication in prokaryotes and eukaryotes

DNA replication occurs ___ is both prokaryotes and eukaryotes.

A

Bi-directionally

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
41
Q

Replication in prokaryotes and eukaryotes

Two replication forks travel in ___ directions, unwinding the helix as they go.

A

Opposite

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
42
Q

Replication in prokaryotes and eukaryotes

In bacteria, there is only one ___ __ ___, both directions till it meets at the other side at the terminus, terC.

A

Origin of replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
43
Q

Replication in prokaryotes and eukaryotes

Halfway through this process, the chromosome looks like the Greek letter theta ->

A

Theta replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
44
Q

Replication in prokaryotes and eukaryotes

Some plasmids and many viruses replicate their genomes by a process called ___ ___ ___.

A

Rolling circle replication

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
45
Q

Replication in prokaryotes and eukaryotes

Rolling circle replication -

A
  • at the ori, one strand of the DNA is nicked and unrolled
  • DNA is synthesized from the origin using the circular strand as a template
  • the dangling strand is removed, ligated to form a circle and finally a second strand is synthesized, resulting in two rings of plasmids
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
46
Q

Replication in prokaryotes and eukaryotes

There are multiple ___.

A

Ori

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
47
Q

Replication in prokaryotes and eukaryotes

The ___ shorten every time the cell replicates.

A

Telomeres

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
48
Q

Replication in prokaryotes and eukaryotes

In some cells, the enzyme ___ can regenerate the telomere by using an RNA template to synthesize the repeats.

A

Telomerase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
49
Q

Replication in prokaryotes and eukaryotes

Replication in eukaryotes occur at a specific point during the ___ ___.

A

Cell cycle

50
Q

Replication in prokaryotes and eukaryotes

___ is a dynamic process.

A

Mitosis

51
Q

In vitro DNA synthesis

Components:

A

Single-stranded template DNA, DNA polymerase, oligonucleotide primer, and a nucleotide precursors

52
Q

In vitro DNA synthesis

Heat or strong base is used to ..

A

Disrupt H-bond

53
Q

In vitro DNA synthesis

___ primer instead of RNA primer.

A

DNA

54
Q

In vitro DNA synthesis

The primer has a sequence ___ to a short region of DNA template.

A

Complementary

55
Q

In vitro DNA synthesis

The template may be cloned into a ___.

A

Vector

56
Q

Chemical synthesis of DNA

DNA synthesis, amplification, and sequencing all derived from..

A

Knowledge of DNA structure and replication

57
Q

Chemical synthesis of DNA

DNA synthesis, amplification, and sequencing

These methods are essential for..

A

Isolating, characterizing, and expressing cloned genes

58
Q

Chemical synthesis of DNA

The first nucleotide is coupled to a bead with ___ ___.

A

Spacer molecule

59
Q

Chemical synthesis of DNA

Detritilation -

A

The 5’-DMT is removed

60
Q

Chemical synthesis of DNA

Coupling -

A

An activated phosphoramidite nucleotide is added to 5’ and of first nucleotide

61
Q

Chemical synthesis of DNA

Capping -

A

All first nucleotides that were not linked with the second nucleotides were capped to prevent further extension

62
Q

Chemical synthesis of DNA

Oxidation -

A

Phosphite triester is converted to phosphodiester

63
Q

Chemical synthesis of DNA

The first nucleotide is linked to a glass bead via a ___ ___ attached to its 3’-OH group.

A

Spacer molecule

64
Q

Chemical synthesis of DNA

Starting complex for the chemical synthesis of DNA strand:

A
  • the initial nucleoside has a protective DMT group attached to the 5’
  • a spacer molecule attached to the 3’ hydroxyl group of the deoxyribose
  • the spacer unit is attached to a solid support, CPG bead
65
Q

Chemical synthesis of DNA

____ for each of the four bases.

A

Phosphoramidites

66
Q

Chemical synthesis of DNA

Phosphoramidites for each of the four bases.

A ___ ___ is attached to the 3’ phosphite group of the nucleoside.

A ___ ___ protects the 3’ phosphite group of the deoxyribose.

___ ___ attached to the 5’ hydroxyl group of the sugar.

A

Diisopropylamine group

B-cyanoethyl groups

DMT group

67
Q

Chemical synthesis of DNA

Detritylation

A
  • the 5’ DMT group is removed from the attached nucleoside by treatment of trichlotoacetic acid (TCA) to yield reactive 5’ hydroxyl group
  • then, column is washed with acetonitrile to remove TCA and then with argon to remove acetonitrile
68
Q

Chemical synthesis of DNA

Activation and coupling

A
  • after TCA, the next prescribed base and tetrazole are introduced simultaneously
  • the tetrazole activates the phosphoramidite so that its 3’ phosphite forms a covalent bond with 5’ hydroxyl group of the initial nucleoside
69
Q

Chemical synthesis of DNA

Capping of unreacted nucleotide

A

If any of the first nucleotides are not coupled to the second nucleotide, these could react with subsequent nucleotide creating and internal deletion if the nucleotide

70
Q

Chemical synthesis of DNA

Capping

A

The available 5’ hydroxyl group of unreacted detritylated nucleosides are acetylated to prevent them from participating in the coupling reaction of the next cycle

71
Q

Chemical synthesis of DNA

Oxidation

A

The phosphite triester is oxidized to a phosphodiester by adding iodine

This stabilizes the nucleotide for further additions

72
Q

___ are the key components for assembling genes.

A

Oligonucleotides

73
Q

Oligonucleotides

The applications are including..

A

Large-scale production of proteins, testing protein function after changing specific codons, and creating nucleotide sequences that encode proteins with novel properties

74
Q

Oligonucleotides

The production of short genes can be accomplished by..

A

Synthesizing the complementary strands and then annealing them

75
Q

Chemical synthesis of complete genes

The gaps are filled and the nicks are sealed with __ __ ___.

A

T4 DNA ligase

76
Q

___ ___ ___ is a process that uses DNA polymerase in an in vitro replication.

A

Polymerase chain reaction (PCR)

77
Q

PCR is a simple method for making multiple copies of a DNA sequences, such as a ___.

A

Gene

78
Q

Polymerase chain reaction

Denaturing -

A

Double-stranded fragments of DNA heated to denature them into single strands

79
Q

Polymerase chain reaction

Annealing -

A

Short primers matching 3’ end of DNA fragments and sufficient quantities of the four dNTPs are added

80
Q

Polymerase chain reaction

Extending -

A

DNA polymerase catalyzes synthesis of new DNA strands

81
Q

Polymerase chain reaction

Second PCR cycle

A

The templates for this are long templates synthesized during the first PCR cycle and the original DNA strands

82
Q

Polymerase chain reaction

Third PCR cycle

A

During the renaturation step, the primer sequences hybridize to complementary regions of original, long-template, and short-template strands

83
Q

Polymerase chain reaction

Thirtieth PCR cycle

A

By the 30th cycle, the population of DNA molecules in a reaction tube consists almost entirely of short strands

84
Q

Polymerase chain reaction

Anneals ___

Amplifies in what direction?

A

Antiparallel, 5’->3’ direction

85
Q

Polymerase chain reaction

How many copies of genes can be made in just 20 PCR cycles?

A

1 million

86
Q

Polymerase chain reaction

PCR has had an enormous impact on ___ ___.

A

Genetic research

87
Q

Polymerase chain reaction

PCR requires a DNA polymerase that survives high heat extracted from hot springs ->

A

Bacterium Thermus aquaticus

88
Q

Polymerase chain reaction

Considerations:

A
  • DNA template
  • primer design
  • DNA polymerase
  • thermo cycling program
  • reaction conditions
  • controls
89
Q

Polymerase chain reaction

How to determine if successful

A

Agarose gel

90
Q

PCR considerations

DNA template

A
  • PCR is a very robust technique
  • DNA preparation is relatively simple
  • target DNA sequences do not have to be isolated from other DNA
  • great care must be taken to avoid contamination of samples in case of forensics study
  • the starting DNA template may be isolated from prokaryotic or eukaryotic organisms, or such exotic organisms as viruses
91
Q

PCR considerations

Primer design

A
  • DNA primers of 18-24 nucleotides are commonly used
  • may also be degenerate at one or more nucleotides to permit amplification of target DNAs of more variable sequence
  • GC content in the range of 40-60%
  • relatively high melting temperature (60-70C)
  • use primary pairs with Tm not more than 5C different
  • the annealing temp. for use in PCR can further be approximated by subtracting 5C from the Tm
  • long stretches of a single nucleotide should be avoided
  • primers should not be self-complementary nor complementary to the other primer used in the PCR reaction
  • restriction enzyme cleavage sites may be added to the 5’ ends of primers to facilitate post-amplification cloning of PCR products
92
Q

Degenerate primers -

A

Mixtures of primers with different nucleotides occurring at the same primer position on different primer molecules

93
Q

PCR considerations

DNA polymerase

A
  • Taq polymerase has no proofreading ability
  • it is known to introduce an incorrect nucleotide for about every 2x10^4 nucleotides
  • DNA polymerases that incorporate a proofreading ability may be used in place of Taq
94
Q

PCR was developed using ___ ___ ___.

A

Taq DNA polymerase

95
Q

PCR considerations

Thermo cycling program

A
  • denaturation; generally 94C for 5 minutes
  • followed by 25-30 repetitive cycles of denaturing, annealing, and extending
  • denaturing; 94C for 30 seconds
  • annealing; 30-65C for 30 seconds
  • extending; 64-75C for 2-5 minutes
96
Q

PCR considerations

Reaction conditions

A
  • nucleotides
  • magnesium
  • salt concentrations
  • number of cycles and temperature profiles
97
Q

PCR considerations

Controls

A
  • many opportunities for contamination
  • aerosol resistant pipette tips
  • omit DNA template and/or one or both primers
  • control reactions with known positive or negative samples
98
Q

___ ___ allows unknown sequences to be amplified by PCR provided that they are located near a known sequence.

A

Inverse PCR

99
Q

Inverse PCR

The DNA is cut with a ___ ___ that cut upstream and downstream.

A

Restriction enzyme

100
Q

Inverse PCR

The linear piece of DNA is..

A

Circularized and then amplified with primers that annealed to the known region

101
Q

Reverse transcriptase PCR

The choice of primer depends on..

A

The starting RNA template and characteristics or knowledge of the target for amplification

102
Q

___ ___ ___ is used to make a cDNA copy of the starting RNA.

A

Enzyme reverse transcriptase

103
Q

__-___ follows the same steps found in standard PCR from a DNA template.

A

RT-PCR

104
Q

PCR in genetic engineering

Primers for PCR can be designed to have non-homologous regions at the 5’ end that contain the..

A

Recognition sequence for a particular restriction enzyme

105
Q

PCR in genetic engineering

The PCR product is digested with the restriction enzyme, this generates..

A

Sticky ends that are compatible with a chosen vector.

106
Q

PCR in genetic engineering

The ___ ___ activity of Taq polymerase adds an extra adenine at the 3’ ends.

A

Terminal transferase

107
Q

PCR in genetic engineering

The __ ___ ___ was designed so that when linearized, it has a single 5’-thymine overhand.

A

TA cloning vector

108
Q

PCR in genetic engineering

___ ___ can be used to link two different gene segments.

A

Overlapping primers

109
Q

PCR in genetic engineering

The ___ is transformed into the host cell, and homologous crossing over occurs.

A

Cassette

110
Q

The ___ ___ will have mismatch in the middle, but the remaining sequences will be complementary.

A

Mutagenic primer

111
Q

DNA sequencing

The function of a gene can often be deduced from its ___ ___.

A

Nucleotide sequence

112
Q

DNA sequencing

A presumptive ___ ___ ___, determined from the nucleotide sequence, can be compared with protein from known genes.

A

Amino acid sequence

113
Q

DNA sequencing

The ___-___ ___ may provide information about the regulation of a gene.

A

Non-coding regions

114
Q

DNA sequencing

The ___ ___ is essential for molecular cloning studies and characterizing gene activity.

A

Sequence information

115
Q

___ ___ techniques uses modified nucleosides with fluorescent tags.

A

DNA sequencing

116
Q

Normal DNA synthesis

A
  • an incoming dNTP base pairs with the complementary nucleotide of the template strand
  • the internucleotide linkage occurs between the 3’ hydroxyl group of the last nucleotide of the growing strand and the alpha-phosphate group of the incoming nucleotide
117
Q

Blocked DNA synthesis

A
  • chain growth is stopped by the addition of a dideoxynucleotide to the end of the growing strand
  • the next incoming nucleotide cannot be formed because there is no 3’ OH group on the dideoxynucleotide sugar
118
Q

Simulated autoradiograph

A
  • each lane of the gel was loaded with the contents of one of the four reaction tubes
  • by convection, the bands of the autograph are read from the bottom to the top
119
Q

Electrophoresis separates strands by length

A
  • color of fluorescent tag indicates type of ddNTP at end of the strand
  • by checking end color of successive strand lengths, the sequence is revealed.
120
Q

Next generation sequencing

The ___ ___ is listed across the top.

A

Reference sequence

121
Q

Next generation sequencing

Read depth -

A

The number of sequences that map a region in the genome

Biotechnology I (14 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions