Mod 3. PCR Flashcards
(51 cards)
1
Q
Terminology
the term for the product of PCR
A
Amplicons
2
Q
Terminology
the DNA segment amplified
A
Target
3
Q
Terminology
a single stranded sequence of nucleotides known as an oligonucleotide
A
Primer
4
Q
is a DNA polymerase that adds nucleotides complementary to those in the unpaired DNA strand onto the annealed primer. It catalyzes the synthesis of new strands of DNA.
A
enzyme
5
Q
What is the Polymerase Chain Reaction?
A
Polymerase Chain Reaction (PCR) is a technique used to selectively amplify a specific segment of DNA, even if it is part of a larger and complex mixture of DNAs, such as a specific exon of a human gene. It functions like a molecular photocopier, producing numerous copies of the target DNA segment for analysis or further use.
- Through repeated cycles of denaturation, annealing, and extension, the “short products” undergo exponential amplification between the two primers, causing the amount of product to double with each cycle.
6
Q
PRINCIPLE
- is a method of rapid
DNA amplification. - In vitro method for they production large amounts of specific
DNA or RNA fragments of defined length and sequence from
small amounts of short oligonucleotide flanking sequences
(primer)
A
Polymerase Chain Reaction
7
Q
Who develop PCR?
A
*Kary Mullis
*Michael Smith
8
Q
Story behind PCR
How did they develop the PCR?
A
The story behind PCR (Polymerase Chain Reaction) began in 1983 when Kary Mullis had the idea while driving through the hills of northern California, inspired by the precision of DNA replication. He excitedly shared his idea with his girlfriend and later struggled to sleep due to the excitement. Initially, his superiors at Cetus Corporation were skeptical, but Mullis successfully demonstrated the technique the following year by amplifying a gene linked to sickle cell mutation. He published his findings in 1985 and filed for patents, sparking a revolution in DNA amplification. Although he only received a $10,000 bonus, the company sold the technology for $300 million. Mullis was later awarded the Nobel Prize in 1993 for his groundbreaking discovery.
9
Q
What are the Two phases of PCR?
A
Screening phase and Amplication phase
9
Q
Two phases of PCR
during the subsequent rounds of PCR the copy number of the desired sequence increases exponentially
A
Amplification phase
9
Q
What is the use of Polymerase Chain Reaction?
A
- A rapid procedure for in vitro
enzymatic amplification of a
specific segment of DNA. - Allows amplification of nucleic
acid sequences both for the
purposes of disease and
pathogen detection and for the
preparation of hybridization
probes and sequencing
templates.
10
Q
PCR: 3 General Steps
A
- The separation of the strands (denaturation)
- Annealing the primer to the template (annealing)
- The extension/elongation of the new strands
(extension or elongation)
11
Q
Two phases of PCR
during the first few cycles the appropriate part of the template is selected by specific binding and extension of theprimers
A
Screening phase
12
Q
3 steps involved in the amplification process
A
*Denaturation
*Annealing
*Extension
These make up one cycle and are repeated over and over again
for, depending on the specific application, from 25 to 40 times.
13
Q
Which of the 3 steps involved in the amplification process
- The template nucleic acid is made single-stranded by exposure to an elevated temperature (92-98ºC).
- DNA molecule has a pair of strands, in the first cycle, the
double-strands DNA is separated into individual strands by
applying heat (at 94˚C). - The high temperature causes the hydrogen bonds between the
bases in two strands of template DNA to break and the two
strands to separate. - This results in two single strands of DNA, which will act as
templates to produce the new strands of DNA. - It is important that the temperature is maintained at this stage
for long enough to ensure that the DNA strands have
separated completely. - This usually takes between 15-30 seconds.
A
Denaturation
14
Q
Which of the 3 steps involved in the amplification process
- The temperature of the reaction is then brought to between
65°C and 72°C in an annealing step, by which the two primers
attach to opposite strands of the denatured template such that their 3’ ends are directed toward each other. - An initiator of the nucleic acid synthesis, primer is attached
to the strand and counter-strand after the temperature is
lowered. - During this stage, the reaction is cooled to 50-65⁰C.
- This enables the primers to attach to a specific location on
the single-stranded template DNA by way of hydrogen
bonding (the exact temperature depends on the melting
temperature of the primers you are using).
A
Annealing
15
Q
Which of the 3 steps involved in the amplification process
- A thermally stable DNA polymerase adds dNTPs onto the 3’
ends of the two annealed primers such that replication occurs
the area between and including the primer annealing sites. - When the temperature is raised, a heat-resistant polymerase
links complementary nucleotides to both primers in such a
way that both individual strands are transformed into two
double strands. - The heat is increased to 72⁰C to enable the new DNA to be
made by a special Taq DNA polymerase enzyme which adds
DNA bases.
A
Extension
16
Q
The amplification phase of PCR: Polymerization
Parameters influencing the reaction mix:
A
- Buffer
- dNTPs (four deoxynucleotides) concentration
- primer concentration
- MgCl2 concentration
- enzyme concentration
- DNA template concentration (~5ng to 10 ng)
17
Q
Template DNA
They amount of template DNA is in they range of Blank to blank ng for plasmid or phage DNA and 0.1 to 1 g for genomic DNA
A
0.01 to 1,
18
Q
Template DNA
To purify DNA they use the?
A
phenol, EDTA or proteinase
19
Q
- An enzyme taken from the heat-loving bacteria. Thermus aquaticus.
- This bacteria normally lives in hot springs so can tolerate temperatures above 80⁰C.
- The bacteria’s DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.
A
Taq Polymerase
19
Q
is the optimum temperature for the Taq polymerase to build the complementary strand.
A
72⁰C
20
Q
True or false
Too much Mg2+ ions result in a low yield PCR product
and too many increase the yield of non-specific
products and promote misincorporation
A
False (Few)
20
Q
2 primers
A
forward primer and a reverse primer.
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* Single strands of DNA sequence that are around 20 to 30 bases in length.
* Designed to be complementary in sequence to short sections of DNA on each end of the sequence to be copied.
* Serve as the starting point for DNA synthesis.
* The polymerase enzyme can only add DNA bases to a double strand of DNA
Primers
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are the building blocks of DNA. They should be added
to a PCR in equimolar amounts and, depending on the specific
application, are used in concentrations ranging from 20 to 200
M each.
* In preparing a series of PCRs, the four dNTPs can be prepared as a master
Deoxynucleoside triphosphates
## Footnote
Four dNTPS (dATP, dCTP, dGTP, and dTTP)
22
What is the use of PCR buffer?
PCR buffer provides a suitable chemical environment for the activity of DNA polymerase, with a pH typically between 8.0 and 9.5, stabilized by Tris-HCl. For Taq DNA polymerase, potassium ions (K⁺) from KCl promote primer annealing, while ammonium sulfate ((NH₄)₂SO₄) may be used instead to destabilize mismatched primer-template base-pairing, enhancing specificity. DNA polymerases are often supplied with optimized PCR buffers to ensure efficient enzyme activity and reliable results.
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The denaturation temperature, usually
94°C
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# TRUE OR FALSE
The ideal annealing temperature must be high enough to enable hybridization between primer and template, but low enough to prevent mismatched hybrids from forming
False (low, high)
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Why is it important to know the Tm (Melting temperature)
to help ensure that an adequate denaturation temperature is chosen for thermal cycling, one that will provide an optimal amplification reaction.
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*is the temperature at which half of the molecule is in double-stranded conformation and half is in single stranded form. It does not matter where along the length of the molecule
the single-stranded or double-stranded regions occur so long as half of they entire molecule is in single-stranded form.
* Refers to either the forward or reverse primer. It is the temperature at which 50% of the primers of one type are annealed to the DNA template.
Tm (melting temperature)
## Footnote
It is referred to as "melting" temperature because at a low temperature, all of the primers will anneal with the template DNA.
-Longer primers and those with high GC content anneal at higher temperatures compared to shorter primers and those with high AT content.
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Why is DNA called a polyanion?
because of the multiple negative charges along
the sugar–phosphate backbone.
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is frequently added to a PCR to enhance the activity of the polymerase.
Potassium chloride (KCI)
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* One primer only per reaction
* Requires optimization for only one primer only
* Time consuming in terms of the number of runs (if more than one primers per sample)
Single PCR
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* Two or more primers per PCR reaction
* Requires optimization for all primers in one reaction
* Reduce no. of runs and reagents
Multiplex PCR
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* ability to reduce the number of reactions needed to
test a sample for different targets helps saving time
and money and makes multiplex systems useful
especially when large sample numbers have to be
screened.
* Primer concentrations must be adjusted to assure an
even amplification of all targeted DNA fragments.
Multiplex PCR
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# PCR run
a unit of power equal to one joule
per second
Power (Watts)
| Power (W) = Current (A) x voltage (V)
32
# PCR run
the amount of electrical charge flowing part a specified circuit point per unit time. It is measured in amperes (A) or milliamperes (mA).
Current (mA)
| Current (A) = power (W) / Voltage (V)
33
# PCR run
a unit of electrical potential
Voltage (Volts)
| Voltage (V) = power (W) / current (A)
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is used to prepare DNA markers and samples for loading on agarose or polyacrylamide gels. It contains two different dyes
(bromophenol blue and xylene cyanol FF) for visual
tracking of DNA migration during electrophoresis.
Loading dye
## Footnote
*The presence of glycerol ensures that the DNA in the
ladder and sample forms a layer at the bottom of the well.
The EDTA included in the solution binds divalent metal
ions and inhibits metal-dependent nucleases.
35
PCR Application
* The polymerase chain reaction (PCR) is used to amplify a specific segment of DNA and has applications in gene cloning, DNA sequencing, mutation detection, and forensics.
* Identify contaminating microorganisms in food
* Diagnose genetic disease
* Map genes to specific chromosome segments
36
# Positive & Negative Control in PCR
is a reaction that is representative of the quality of our work
and reagents. It is a reaction that has been previously established by another lab and provides us with well characterized results. Therefore, if an experiment or
procedure is prepared without errors, and fresh reagents are used, we expect a specific result from a positive control. When such a result is not obtained, it is necessary to re-evaluate our technique, calculations, and reagent quality.
Positive control
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# Positive and Negative Control in PCR
must always be performed when setting up PCR reactions. A
PCR product obtained during a reaction may not always be the desired product. A primary concern in PCR is contamination of the reaction with foreign, unwanted DNA. We all carry some DNA on our hands, and it is important to wear gloves when handling PCR reagents to prevent contamination of these reagents. In our lab, there is a designated area with specific pipettes that are to be used in PCR preparation only. One reason for these precautions is that contaminant DNA may act as a template for the PCR primers, resulting in unwanted PCR
products. Another reason for these precautions is to avoid any enzymes that may cut up your template DNA
Negative control
## Footnote
The simplest way to rule out that your PCR product may be an amplification of contaminant DNA is to prepare a negative control. The negative control
contains all the reagents as your other PCR tubes (0.25 μM of each primer, 1X
PCR buffer, 1.5mM MgCl2, 0.25mM dNTPs, Taq DNA polymerase, and water),
EXCEPT template DNA.
38
# Other Types of Polymerase Chain Reaction
¨ a way of quantifying the amplification of DNA as it occurs realtime
¨ Uses fluorescent dyes that yield increasing fluorescent signal indirect
A real-time PCR (rt-PCR)
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# Other Types of Polymerase Chain Reaction
¨ starts with the reverse transcription of either total RNA
or polyA+ RNA (mRNA) into cDNA using a reverse
transcriptase
¨ next, approximately 10% of the cDNA is transferred toa
separate tube for the real timePCR reaction
qPCR (quantitative reverse transcriptionPCR)
40
# Other Types of Polymerase Chain Reaction
offers a logistically simpler protocol: a relatively rapid DNA amplification reaction occurs at one temperature, and the products are visualized with a colour change within the reaction tubes.
* first developed over 20 years ago (Notomi et al. 2000)
* uses a single enzyme, a DNA polymerase with strand
displacement activity that is able to massively amplify from only a few copies of DNA. The amplification protocol requires only a single temperature for the reaction (which can be provided by a thermoblock), and amplification success is diagnosed without the need for electrophoretic techniques.
LAMP (loop-mediated isothermal amplification)
| B. Lamp
41
# Nucleic Acid Stains used in Agarose Gel Electrophoresis
is used as a stain for DNA in agarose gels. The ethidium bromide molecule intercalates (inserts) between the stacked bases of double-stranded DNA. The intercalated Blank molecules emit fluorescent light when irradiated with ultraviolet light. Three hundred nanometers is the optimal wavelength for exciting fluorescence from the ethidium bromide-DNA complex. Excitation at 254 nm gives only one third as much fluorescence, while excitation at 366 nm yields
only one seventh as much fluorescence.
Ethidium Bromide
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is a promising alternative to poly- merase chain reaction (PCR) for rapid SARS-CoV-2 testing, including methods leveraging extraction-free amplification
Reverse transcription (RT)-LAMP
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# Nucleic Acid Stains used in Agarose Gel Electrophoresis
are next-generation fluorescent nucleic acid gel
stains designed to replace the highly toxic ethidium bromide (EtBr).
GelRed and GelGreen
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# Nucleic Acid Stains used in Agarose Gel Electrophoresis
* are highly sensitive reagents for staining DNA in electrophoresis gels. These gel stains provide greater
sensitivity, with lower background fluorescence, than the conventional ethidium bromide gel stain.
* exhibited very low mutagenicity compared to ethidium bromide, when tested by an independent, licensed
testing laboratory, and it is not classified as hazardous waste or as a pollutant under U.S. federal regulations.
Molecular Probes® fluorescent nucleic
acid gel stains
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As a future biotechnologist, how would you design a cloning strategy for a PCR product with blunt ends versus one with sticky ends?
As a future biotechnologist, I would design a cloning strategy based on the type of PCR product. For blunt-ended products, I would use a blunt-ended vector for ligation with T4 DNA ligase or attach adaptors to create sticky ends for more efficient insertion. For sticky-ended products, I would take advantage of the adenine overhang added by Taq polymerase by using a vector with thymidine overhangs or design primers with restriction sites to generate cohesive ends for efficient ligation. This approach would ensure successful and accurate PCR product cloning.
