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
2
Q
Why is an RNA primer necessary during replication?
A
DNA polymerase III requires a 3’-OH to elongate DNA
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
4
Q
Which of the following statements about mismatch repair is incorrect?
A
MutSHL can synthesize new DNA after a mismatch has been excised
5
Q
Which of the following statements is incorrect regarding DNA replication?
A
Prokaryotic chromosomes have multiple origins of replication
6
Q
During in vitro DNA replication, which of the following components is not required?
A
DNA helicase to separate the strands
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
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.
9
Q
Which of the following components terminates the chain in a sequencing reaction?
A
Dideoxynucleotides
10
Q
Which of the following statements about PCR is incorrect?
A
The DNA template is denatured using helicase
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
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
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
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
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
16
Q
In ___ sequencing, the DNA fragments are found to a solid surface via a flow cell
A
Illumina
17
Q
Flashes of light are emitted whenever a base is added in ___ sequencing.
A
454
18
Q
Replication starts at an ___ __ ___.
A
Origin of replication (ori)
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
20
Q
Semiconservative replication -
A
Each copy has one strand from the original helix and one new strand
21
Q
Replication of DNA
DNA ___ removes the supercoiling.
___ unwinds the double helix.
A
Gyrase; helicase
22
Q
Replication of DNA
___-___ ___ ___ coats the single-stranded regions.
A
Single-stranded binding protein
23
Q
Replication of DNA
DNA ___ links the 3’-OH and 5’-PO4 of neighboring nucleotides, forming ___ ___.
A
Ligase; phosphodiester bond
24
Q
Replication of DNA
The final step is to add ___ ___ along the new strand.
A
Methyl groups
25
Replication of DNA
There is a delay in methylating the new strand, thus, the DNA double helix is ___.
Hemimethylated
26
Replication of DNA
___ ___ and ___ ___ add the methyl groups onto the newly synthesized DNA strand.
DAM methylase and DCM methylase
27
Replication of DNA
The two daughter copies of circular chromosomes may become ___, or connected like two links of chain.
Catenated
28
Replication of DNA
___ __ untangles the two chromosomes so they can partition into the daughter cells.
Topoisomerase IV
29
Replication of DNA
DNA polymerase cannot synthesize new DNA without a pre-existing ___.
3'-OH
30
Replication of DNA
DNA replication requires an ___ ___ to initiate strand formation.
RNA primer
31
Replication of DNA
The ___ protein displaces the ___ proteins.
PriA; SSB
32
Replication of DNA
___ associates with the PriA protein.
Primase
33
Replication of DNA
The ___ makes the short DNA primer.
Primase
34
Replication of DNA
The ___ ___ is composed of alternating Okazaki fragments and RNA primers.
Lagging strand
35
Replication of DNA
___ ___ binds to the primer region, and as it moves forward, it degrades the RNA and replaces it with DNA.
DNA polymerase I
36
Replication of DNA
___ ___ seal the nick in the phosphate backbone.
DNA ligase
37
Replication of DNA
Unwinds in one direction, synthesizes in opposite direction
Lagging strand
38
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.
Mismatch repair proteins (MutSHL); DNA polymerase III
39
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.
MutS protein
MutH
MutL
DNA polymerase III
40
Replication in prokaryotes and eukaryotes
DNA replication occurs ___ is both prokaryotes and eukaryotes.
Bi-directionally
41
Replication in prokaryotes and eukaryotes
Two replication forks travel in ___ directions, unwinding the helix as they go.
Opposite
42
Replication in prokaryotes and eukaryotes
In bacteria, there is only one ___ __ ___, both directions till it meets at the other side at the terminus, terC.
Origin of replication
43
Replication in prokaryotes and eukaryotes
Halfway through this process, the chromosome looks like the Greek letter theta ->
Theta replication
44
Replication in prokaryotes and eukaryotes
Some plasmids and many viruses replicate their genomes by a process called ___ ___ ___.
Rolling circle replication
45
Replication in prokaryotes and eukaryotes
Rolling circle replication -
- 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
46
Replication in prokaryotes and eukaryotes
There are multiple ___.
Ori
47
Replication in prokaryotes and eukaryotes
The ___ shorten every time the cell replicates.
Telomeres
48
Replication in prokaryotes and eukaryotes
In some cells, the enzyme ___ can regenerate the telomere by using an RNA template to synthesize the repeats.
Telomerase
49
Replication in prokaryotes and eukaryotes
Replication in eukaryotes occur at a specific point during the ___ ___.
Cell cycle
50
Replication in prokaryotes and eukaryotes
___ is a dynamic process.
Mitosis
51
In vitro DNA synthesis
Components:
Single-stranded template DNA, DNA polymerase, oligonucleotide primer, and a nucleotide precursors
52
In vitro DNA synthesis
Heat or strong base is used to ..
Disrupt H-bond
53
In vitro DNA synthesis
___ primer instead of RNA primer.
DNA
54
In vitro DNA synthesis
The primer has a sequence ___ to a short region of DNA template.
Complementary
55
In vitro DNA synthesis
The template may be cloned into a ___.
Vector
56
Chemical synthesis of DNA
DNA synthesis, amplification, and sequencing all derived from..
Knowledge of DNA structure and replication
57
Chemical synthesis of DNA
DNA synthesis, amplification, and sequencing
These methods are essential for..
Isolating, characterizing, and expressing cloned genes
58
Chemical synthesis of DNA
The first nucleotide is coupled to a bead with ___ ___.
Spacer molecule
59
Chemical synthesis of DNA
Detritilation -
The 5'-DMT is removed
60
Chemical synthesis of DNA
Coupling -
An activated phosphoramidite nucleotide is added to 5' and of first nucleotide
61
Chemical synthesis of DNA
Capping -
All first nucleotides that were not linked with the second nucleotides were capped to prevent further extension
62
Chemical synthesis of DNA
Oxidation -
Phosphite triester is converted to phosphodiester
63
Chemical synthesis of DNA
The first nucleotide is linked to a glass bead via a ___ ___ attached to its 3'-OH group.
Spacer molecule
64
Chemical synthesis of DNA
Starting complex for the chemical synthesis of DNA strand:
- 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
Chemical synthesis of DNA
____ for each of the four bases.
Phosphoramidites
66
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.
Diisopropylamine group
B-cyanoethyl groups
DMT group
67
Chemical synthesis of DNA
Detritylation
- 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
Chemical synthesis of DNA
Activation and coupling
- 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
Chemical synthesis of DNA
Capping of unreacted nucleotide
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
Chemical synthesis of DNA
Capping
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
Chemical synthesis of DNA
Oxidation
The phosphite triester is oxidized to a phosphodiester by adding iodine
This stabilizes the nucleotide for further additions
72
___ are the key components for assembling genes.
Oligonucleotides
73
Oligonucleotides
The applications are including..
Large-scale production of proteins, testing protein function after changing specific codons, and creating nucleotide sequences that encode proteins with novel properties
74
Oligonucleotides
The production of short genes can be accomplished by..
Synthesizing the complementary strands and then annealing them
75
Chemical synthesis of complete genes
The gaps are filled and the nicks are sealed with __ __ ___.
T4 DNA ligase
76
___ ___ ___ is a process that uses DNA polymerase in an in vitro replication.
Polymerase chain reaction (PCR)
77
PCR is a simple method for making multiple copies of a DNA sequences, such as a ___.
Gene
78
Polymerase chain reaction
Denaturing -
Double-stranded fragments of DNA heated to denature them into single strands
79
Polymerase chain reaction
Annealing -
Short primers matching 3' end of DNA fragments and sufficient quantities of the four dNTPs are added
80
Polymerase chain reaction
Extending -
DNA polymerase catalyzes synthesis of new DNA strands
81
Polymerase chain reaction
Second PCR cycle
The templates for this are long templates synthesized during the first PCR cycle and the original DNA strands
82
Polymerase chain reaction
Third PCR cycle
During the renaturation step, the primer sequences hybridize to complementary regions of original, long-template, and short-template strands
83
Polymerase chain reaction
Thirtieth PCR cycle
By the 30th cycle, the population of DNA molecules in a reaction tube consists almost entirely of short strands
84
Polymerase chain reaction
Anneals ___
Amplifies in what direction?
Antiparallel, 5'->3' direction
85
Polymerase chain reaction
How many copies of genes can be made in just 20 PCR cycles?
1 million
86
Polymerase chain reaction
PCR has had an enormous impact on ___ ___.
Genetic research
87
Polymerase chain reaction
PCR requires a DNA polymerase that survives high heat extracted from hot springs ->
Bacterium Thermus aquaticus
88
Polymerase chain reaction
Considerations:
- DNA template
- primer design
- DNA polymerase
- thermo cycling program
- reaction conditions
- controls
89
Polymerase chain reaction
How to determine if successful
Agarose gel
90
PCR considerations
DNA template
- 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
PCR considerations
Primer design
- 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
Degenerate primers -
Mixtures of primers with different nucleotides occurring at the same primer position on different primer molecules
93
PCR considerations
DNA polymerase
- 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
PCR was developed using ___ ___ ___.
Taq DNA polymerase
95
PCR considerations
Thermo cycling program
- 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
PCR considerations
Reaction conditions
- nucleotides
- magnesium
- salt concentrations
- number of cycles and temperature profiles
97
PCR considerations
Controls
- 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
___ ___ allows unknown sequences to be amplified by PCR provided that they are located near a known sequence.
Inverse PCR
99
Inverse PCR
The DNA is cut with a ___ ___ that cut upstream and downstream.
Restriction enzyme
100
Inverse PCR
The linear piece of DNA is..
Circularized and then amplified with primers that annealed to the known region
101
Reverse transcriptase PCR
The choice of primer depends on..
The starting RNA template and characteristics or knowledge of the target for amplification
102
___ ___ ___ is used to make a cDNA copy of the starting RNA.
Enzyme reverse transcriptase
103
__-___ follows the same steps found in standard PCR from a DNA template.
RT-PCR
104
PCR in genetic engineering
Primers for PCR can be designed to have non-homologous regions at the 5' end that contain the..
Recognition sequence for a particular restriction enzyme
105
PCR in genetic engineering
The PCR product is digested with the restriction enzyme, this generates..
Sticky ends that are compatible with a chosen vector.
106
PCR in genetic engineering
The ___ ___ activity of Taq polymerase adds an extra adenine at the 3' ends.
Terminal transferase
107
PCR in genetic engineering
The __ ___ ___ was designed so that when linearized, it has a single 5'-thymine overhand.
TA cloning vector
108
PCR in genetic engineering
___ ___ can be used to link two different gene segments.
Overlapping primers
109
PCR in genetic engineering
The ___ is transformed into the host cell, and homologous crossing over occurs.
Cassette
110
The ___ ___ will have mismatch in the middle, but the remaining sequences will be complementary.
Mutagenic primer
111
DNA sequencing
The function of a gene can often be deduced from its ___ ___.
Nucleotide sequence
112
DNA sequencing
A presumptive ___ ___ ___, determined from the nucleotide sequence, can be compared with protein from known genes.
Amino acid sequence
113
DNA sequencing
The ___-___ ___ may provide information about the regulation of a gene.
Non-coding regions
114
DNA sequencing
The ___ ___ is essential for molecular cloning studies and characterizing gene activity.
Sequence information
115
___ ___ techniques uses modified nucleosides with fluorescent tags.
DNA sequencing
116
Normal DNA synthesis
- 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
Blocked DNA synthesis
- 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
Simulated autoradiograph
- 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
Electrophoresis separates strands by length
- 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
Next generation sequencing
The ___ ___ is listed across the top.
Reference sequence
121
Next generation sequencing
Read depth -
The number of sequences that map a region in the genome
