Activity 3: DNA (i.e., gfp gene) Amplification by Polymerase Chain Reaction (PCR) Flashcards
(104 cards)
1
Q
Experimental technique that enables the replication of short DNA molecules in vitro or outside the cell
A
Polymerase chain reaction (PCR)
2
Q
→ Since PCR performs DNA replication outside the cell, enzymes that normally conduct the DNA replication process are not available
what are used to make up for the absence of these enzymes?
A
Cycling of high temperatures and a thermostable DNA polymerase
3
Q
are designed to replicate a target DNA region
A
DNA primers
3
Q
where does the Primer attach to?
A
bind to the region of the template strand (at the 3’ end) before the target DNA region
4
Q
are short DNA fragments (Note: RNA fragments are utilized in the natural DNA replication inside cells) that bind to the region of the template strand (at the 3’ end) before the target DNA region
A
Primers
4
Q
In a PCR experiment, how many DNA primers are used?
A
2
one for each template strand;
1 double-stranded DNA is made up of 2 template strands
4
Q
Polymerase chain reaction (PCR)
→ Temperature cycling is done using a
A
thermal cycler
4
Q
→ 1 PCR cycle temperatures
Denaturing phase
Annealing phase
Extension phase
A
94-96°C
~55°C, depending on primer sequence
72°C
4
Q
A co-factor of Taq polymerase, ensures optimal activity of Taq polymerase
A
Mg2+
4
Q
Source of Mg2+
A
MgCl2
4
Q
To successfully make sufficient copies of target DNA, samples are typically run through at least ____ PCR cycles
A
30
4
Q
Central dogma of molecular biology
A
DNA → RNA → Protein
4
Q
Theory stating that genetic or heritable information in biological systems flows in only one direction:
Segments of DNA, called genes, are transcribed into mRNA. mRNA is then “read” in three-nucleotide codons to specify the order of amino acids in a protein.
A
Central dogma of molecular biology
4
Q
The difference between the ribose found in RNA and the deoxyribose found in DNA
A
is that ribose has a hydroxyl group at the 2 carbon
4
Q
Five-carbon sugar (Ribose) of RNA nucleotide has a __________ attached to its 2nd carbon
A
hydroxyl (OH) group
4
Q
Five-carbon sugar (Deoxyribose) of DNA nucleotide has __________ at the 2nd carbon
A
no hydroxyl group
4
Q
Each DNA nucleotide is made up of
A
a sugar, a phosphate group, and a base.
4
Q
The four DNA nucleotides are
A
Adenine. Thymine, Guanine, and Cytosine.
4
Q
The nucleotides are grouped into 2 according to the structure of the nitrogenous base.
A
purines
Pyrimidines
4
Q
Purines
A
Guanine & Adenine
4
Q
Pyrimidines
A
Cytosine, Uracil (in RNA), & Thymine
4
Q
→ A DNA strand is formed by the sugar-phosphate groups of the
A
nucleotides
4
Q
→ is made up of two strands that are held together by hydrogen bonding between bases of the nucleotides located in the same position of both strands
A
DNA double helix
4
Q
→ A purine pairs with a pyrimidine: how many hydrogen bonds each pair?
A
A-T (2 hydrogen bonds)
G-C (3 hydrogen bonds)
4
→ Strands of the DNA double helix are ______- parallel but opposite directionality
anti-parallel
4
explain anti-parallel
* Starting at one end, first/last nucleotide of one strand has an unbound phosphate attached to the 5th sugar carbon and first/last nucleotide of the other strand has an unbound 3rd sugar carbon
4
DNA strand is formed by the addition of
nucleotides at the 3' end of the strand
4
is formed between the phosphate (attached to the 5th sugar carbon) of an incoming nucleotide and the hydroxyl (OH) group at the 3rd sugar carbon of the last nucleotide of a growing DNA strand
phosphodiester bond
4
DNA replication is __________X , meaning each new DNA molecule is composed of one original strand and one newly synthesized strand. This ensures that each daughter cell inherits a complete and accurate copy of the genetic information.
semiconservative
4
Sequence of new DNA strand is NOT the same as BUT complementary to sequence of its
template strand
4
→ The 2 strands of the DNA double helix separate at each origin of replication, forming a
replication bubble
5
→ DNA replication (synthesis of new DNA strands) proceeds at ___________ (replication forks) of the replication bubble
opposite ends
5
is a protein that binds to the double helix ahead of the replication fork and relieves the strain placed on the double helix as it unravels.
Topoisomerase
5
synthesizes the RNA primer on-site- the RNA primer will already by bound to the complementary region of the template strand
Primase
5
breaks, swivels, and rejoins the parental DNA ahead of the replication fork, relieving the strain caused by unwinding.
Topoisomerase
5
unwinds and separates the parental DNA strands.
Helicase
5
proteins that stabilize the un- wound parental strands.
Single-strand binding proteins
5
synthesizes RNA primers, using the parental DNA as a template.
Primase
5
breaks the hydrogen bonds between the two strands. This allows the DNA to unwind into two strands at the replication fork.
DNA helicase
5
prevent the separated strands from joining together again by binding to the separated strands and stabilizing them.
Single-strand binding proteins
6
Helicase
Unwinds parental double helix at replication forks
6
Single stranded binding protein
Binds to and stabilizes single- stranded DNA until it is used as a template
7
topoisomerase
Relieves overwinding strain ahead of replication forks by breaking. swiveling, and rejoining DNA strands
7
primase
Synthesizes an RNA primer at 5' end of leading strand and at 5 end of each Okazaki fragment of lagging strand
7
DNA pol III
Using parental DNA as a template, synthesizes new DNA strand by adding nucleotides to an RNA primer or a pre-existing DNA strand
8
DNA pol I
Removes RNA nucleotides of primer from 5' end and replaces them with DNA nucleotides added to 3' end of adjacent fragment
9
helicase is replaced by
→ Replaced by 95 ̊C (Denaturing temperature in PCR)
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single stranded binding proteins replaced by
→ Replaced by 95°C
11
topoisomerase replaced by
→ Replaced by 95°C; in natural DNA replication, the 2 strands gradually separate, BUT in PCR, high temp. completely separates the 2 strands
12
primase replaced by
→ DNA primers are provided in PCR; also,
annealing temperature allows primers to bind to the complementary region of the template strand
13
DNA pol III replaced by
→ Replaced by Taq polymerase, a thermostable DNA polymerase**
13
DNA pol I replaced by
→ No need, since DNA primers are used in PCR
14
→ Designed (or selected) primers should be _____________ to the target gene
complementary to the regions of the template strands adjacent
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Rules for primer design
primers should bind at the _______ of the gene of interest
flanking region
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refers to the DNA sequences located on either side of a specific gene or other DNA sequence of interest.
These regions play a crucial role in regulating gene expression and other cellular processes.
flanking region
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✔Primers should be ___________ bp long
18-25
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✔Primer melting temperature should be within the range of ________ secondary structures
50-60-C
18
→ If primer is too long, it will be prone to _______, which will prevent it from binding to the template strand
folding
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→ If primer is too short, its sequence will not be very specific, and it will be more prone to binding to incorrect regions of the template strand
this phenomenon is called
(Non-specific binding)
20
Melting temperature is not only used as basis for determining annealing temp., which is the actual temperature used for the annealing stage of the PCR cycle, but ______________ indicates that the primer may be too long and/or has high number of G and C nucleotides
very high Tm
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Tm formula
Tm=4°C(# of GC bp) +2°C (# of AT bp)
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If the annealing temperature is too low:
nonspecific binding may occur.
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If the annealing temperature istoo high:
primers may not bind efficiently.
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+Primer annealing temperature is dependent on
primer Tm
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Temperature at which 50% of primer strands are double- stranded (bound to template DNA)
Primer melting temperature (Tm)
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o If annealing temp. is set __________, accuracy of primer binding increases, but fewer primers bind to the template DNA
higher than Tm
25
temperature used during PCR is set a few degrees below the melting
temperature
→Annealing temperature
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Tm of the 2 primers should NOT have a difference greater than
5°C
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o If annealing temp. _________, more primers will bind to the template, but likelihood of non-specific binding/incorrect base-pairing also increases
is set much lower than Tm
28
Determining theoretical number of PCR products
→ If there is more than 1 copy of the target DNA region (double stranded DNA) before PCR begins, use the following formula to calculate the theoretical number of copies after n PCR cycles:
Theoretical number of double stranded copies of target DNA region after PCR = x* 2^(n)
(x is initial number of double stranded target DNA region; n is number of PCR cycles)
29
→ If there is more than 1 copy of the target DNA region (double stranded DNA) before PCR begins, use the following formula:
PCR = x*2^(n+1)
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Theoretical number of single stranded copies of target DNA region after PCR is
Theoretical number of single stranded copies of target DNA region after PCR = 2^(n+1)
30
→ If there is only 1 copy of the target DNA region (double stranded DNA) before the cycling starts, after one PCR cycle, there will be 2 double stranded copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies, and so on.
true
31
→ Length of the PCR product is from
the first nucleotide of the forward primer to the first nucleotide of the reverse primer
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example:
find the number of nucleotides between nucleotide 134 and 25
110 nucelotides
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After Cycle 1
→ Product for PCR Cycle 1 can be slightly longer than the actual PCR product if time for extension phase is
extended
34
→ But after PCR Cycle 1, even if time for extension phase per cycle is extended, the PCR product will be the sequence between the
2 primers
35
Application of PCR in genetic engineering
→ It can be used to verify if the foreign gene was inserted into the_______
plasmid vector
36
Application of PCR in genetic engineering
→ how can it can be used to verify if the foreign gene was inserted into the plasmid vector?
o After cutting the foreign gene and the plasmid vector with the same restriction enzyme(s), we mix them in one solution, BUT not all plasmids will be inserted with the foreign gene
o Later, bacteria will be exposed to the plasmids; all the plasmid vectors have the antibiotic resistance gene, so a bacterial cell can still grow in the culture medium with antibiotic even if it acquired a plasmid without the foreign gene
37
Application of PCR in genetic engineering
→ It can be used to make many copies of the foreign gene for insertion later into other plasmid vectors
true
38
Not all plasmid vectors will successfully be inserted with the foreign gene
true
38
→ PCR can be used to check which bacterial cell/colony got the plasmid containing the foreign gene
true
38
* All bacterial cells that acquired the plasmid vector, ________, will grow on the culture plate with antibiotic
with or without the foreign gene
39
Mg2+ also binds to negatively charged nucleotides and nucleic acids; why?
Consequently, Mg2+ neutralizes the negative charge of nucleotides and nucleic acids, and this promotes the binding of DNA strands to each other
39
A co-factor of Taq polymerase, _________ binds to the active site of the enzyme and facilitates the addition of new nucleotides to the growing DNA strand
Mg2+
40
Using a higher than optimal concentration of Mg2+ in PCR will promote the binding of the primer to incorrect regions of the template, how does this happen?
this results to?
resulting in non-specific PCR products, since charge neutralization by Mg2+ allows DNA strands to more easily bind to each other
41
Adding too much dNTPs in the PCR solution will result in
Mg2+ depletion
41
→Biosafety cabinet Has ___________ filters in the exhaust system, to effectively trap all known infectious agents and ensure that only microbe-free exhaust air is discharged back into the room and into the work area of the cabinet
high-efficiency particulate air (HEPA)
42
Adding too much dNTPs in the PCR solution will result in Mg2+ depletion, how?
since Mg2+ can also bind to individual nucleotides; Consequently, fewer Mg2+ will be available for the Taq polymerase, and if the number of active enzymes decreases, fewer copies of the target gene will be produce
42
→ Used for working with infectious biological agents
→ Protects the user, the environment, and the material/sample
Biosafety cabinet
43
biosafety cabinet Have different classifications:
Class I
Class II
Class III
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can remove at least 99.97% of particles whose diameter is 0.3 microns (um); its filtration efficiency is higher for particles whose diameter is smaller or larger than 0.3 μm
→ HEPA filters
45
HEPA filters
can remove at least 99.97% of particles whose diameter is
0.3 μm
46
→ Most frequently used biosafety cabinet
Class II Biosafety Cabinet
47
→ Used for working with hazardous chemicals that tend to generate fumes, vapors, and particulate matter
→ Protects the user
→ Does NOT have HEPA filters
→ Air inside the hood is exhausted, without filtration, outside the laboratory
Chemical fume hood
48
Class II Biosafety Cabinet
explain how unfiltered air is circulated
→ Unfiltered air from the room is drawn towards the grille at the front of the work surface, preventing its contact with the sample/material; the air
goes underneath the work surface and proceeds to the back and then to the top of the cabinet where it passes through the HEPA filters
49
Class II Biosafety Cabinet
how does the filtered air travel?
→ A portion of the filtered air is blown downward into the work area of the cabinet; some filtered air is also released back into the room or expelled from the room/building
49
Class II Biosafety Cabinet
→ Filtered air blown into the work area of the cabinet and exposed to the material/sample will be pulled towards the grille at the front and back of the work surface, why?
preventing the user from being exposed to this potentially contaminated air
50
→ Completely enclosed, ventilated cabinet
→ Work inside the cabinet is done with the use of attached rubber gloves → Provides maximum user, environment, and material/sample protection
Class III Biosafety Cabinet
50
Class III Biosafety Cabinet
→ Also known as
"glove box"
51
→ Used for working with non-infectious material, such as culture media preparation and plant tissue culture
→ Intended to protect the material/product, not the user or the environment
Laminar flow hood
51
Laminar flow hood, Direction of airflow
→ is towards the user
52
Laminar flow hood
→ Air goes through the HEPA filter __________ entering the hood
before
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→ Used for working with hazardous chemicals that tend to generate fumes, vapors, and particulate matter
→ Protects the user
→ Does NOT have HEPA filters
→ Air inside the hood is exhausted, without filtration, outside the laboratory
Chemical fume hood
