[LAB] Unit 4.2_The PCR Reaction Chemistry Flashcards
DNA denatures at what temperature?
90+ °C
- PCR is only made possible due to the discovery of this polymerase
-It is stable enough to withstand 90+ °C
Taq polymerase
Genus and specie of this thermostable bacteria
Thermus aquaticus (TAQ)
What are the PCR reaction components? (7)
-Water
-Buffer
-DNA template
-Primers
-Nucleotides
-Mg++ ions
-DNA polymerase
Do Not Play While Being MD
This PCR reaction component is:
-The medium for all other components
-It is specially purified, double-distilled, deionized, autoclaved, and nuclease-free
Water
“PCR-grade Water” yung label sa picture ng bottle
(T/F): We can also use Triple Distilled Water
True
This PCR reaction component:
-contains region to be amplified
-any DNA desired
DNA template
(T/F) Purity is required for DNA template
FALSE. Purity is NOT required.
However, there are instances wherein protein might interfere with your runs into quenching
Increase quenching = decreased in fluorescence
(T/F) Increase quenching = increase in fluorescence
FALSE.
Increase quenching = decreased in fluorescence
(T/F) Water should not contain detectable amounts of nucleic acids
True
This PCR reaction component:
- stabilizes the DNA polymerase, DNA, and nucleotides
Buffer
Examples of Buffer used in PCR
- 500 mM KCl
- 100 mM Tris-HCl, ph 8.3
- Triton X-100 or Tween
(T/F) In the context of Buffer, optimum pH would mean that your enzymes will work correctly
True
This PCR reaction component:
-specific ends of amplified region
-annealing temps should be known depending on its:
*length,
*GC content,
etc.
-This would tell you when to start and end your PCR
Primers
Length of Primer
15-30 nt
Concentration of Primer
0.1 - 1.0 uM (pMol/ul)
What are the two (2) types of Primers?
Forward and Reverse primers
- This primer will tell you when to begin replicating your DNA molecule; when to begin your extension
-from 5 region to 3 region
Forward primer
-This primer tells you where to stop; where to end your extension
-nakabaliktad (3’ to 5’)
Reverse primer
(T/F) the sequence in between the forward and reverse primers are the only ones that will replicate
True
-Has a marker which helps you detect formation of the DNA segment you need
Probe
This PCR reaction component:
- is added to the growing chain
- Activated NTP’S
–dATP, dGTP, dCTP, dTTP
Nucleotides
Storage condition of Nucleotides
Stored at 10mM, ph 7.0
How much Nucleotides is added in assay?
20-200 uM
These are the building blocks
Nucleotides
Nucleotides is in in the form of ___________
dinucleotide triphosphate (dNTPs)
4 types of dNTPs
Adenine
Guanine
Cytosine
Thymine
This PCR reaction component:
-is an Essential co-factor of DNA Polymerase
Magnesium
This PCR reaction component:
-the reason why EDTA is used
Magnesium
note: EDTA creates ions like calcium and magnesium from the blood sample via chelation
This PCR reaction component:
- Stabilizes the DNA double-helix
Magnesium
Too little of Mg++ ions:
a. Enzymes won’t work
b. non-specific priming
c. Band smearing
a. Enzymes won’t work
Too much Mg++ ions:
a. will make DNA extra stable
b. Non-specific priming
c. Band smearing
d. All of the above
d. All of the above
a. will make DNA extra stable
b. Non-specific priming
c. Band smearing
Concentration of Magnesium used in the assay
0.5 - 3.5 uM
This PCR reaction component:
- the enzyme that does the extension
DNA polymerase
This PCR reaction component:
- TAQ or similar
- Heat stable
DNA polymerase
(T/F) In mixing common reagents, add additional 1-2x over and above the required volume
True.
To compensate for pipetting errors
What is next after the last step in mixing common reagents?
Adding the DNA
This happens when the DNA molecules splits into two
Denaturation
This happens when the primer starts binding to the DNA
Annealing
This device gives an environment wherein your sample can encounter temperatures that allow for denaturing, annealing, and extension
Thermal cycler
This happens when the DNA polymerase starts adding nucleotide bases
Extension
(T/F) The copy of your DNA products are independent on the number of cycles
False.
Dependent
How to estimate the number of molecules in a PCR run?
2ⁿ
2 = constant
and raise it by the power of n
n = number of cycles
You subjected your PCR run in 30 cycles.
what is your estimate number of copies?
at least 2³⁰ copies
(T/F)
The more you subject your enzymes to cycling temperatures, the less effective they become.
True
(T/F)
If you subject your Taq polymerase enzyme to repeated temperature variations, its effectivity would not be affected.
False.
Like most things, heat will eventually degrade things over time.
If you subject your Taq polymerase enzyme to repeated temperature variations, mawawala yung effectivity ng enzymes.
(T/F)
PCR run is dependent on the amount of dnTPs that you put in
True.
You can’t build a skyscraper with building blocks only enough for a bungalow
Visualizing results:
After thermal cycling, tubes are taken out of the PCR machine. The contents of the tubes are loaded onto an _______ ___
Agarose gel
Visualizing results:
PCR products are visible as different “_____s”
Bands
What is the end product of a PCR run?
Amplicon
After a cycle run, what is left in your reaction mix is your _______
Amplicon
Modification to Traditional PCR
(3)
Nested PCR
Multiplex PCR
Digital PCR
This PCR occurs when you amplify a large region of your DNA molecule, then once you are able to replicate that segment, you will do another PCR of a smaller section of that fragment.
Nested PCR
What is the major concern for Nested PCR?
contamination that occurs during the transfer of the first-round product to the second tube for the second round of amplification
This PCR was developed to increase both the sensitivity and specificity of PCR
Nested PCR
This technique uses two pairs of amplification primers and two rounds of PCR
Nested PCR
In this PCR, multiple primer sets designed for amplification of different targets are included in the same PCR reaction
(2 or more primer sets used in one PCR run)
Multiplex PCR
Example of usage for Nested PCR
Determining the hair color
What is the prevention step for the major concern for Nested PCR?
by physically separating the first- and second-round amplification mixtures with a layer of wax oil.
Where is Nested PCR used?
a. genome polymorphisms
b. cloning
c. methylation analysis
a. Genome Polymorphisms
-Insertions
-Deletions
-SNPs
Example of use of Multiplex PCR
PCR for HIV
If there are 3 HIV-related genes that we want to test from the patient, just load 1 PCR run with 3 primer sets
(T/F) Multiplex PCR is practical and can’t not save time and effort
True
it can’t not = it can
(T/F) In Multiplex PCR, we can use a set of primers as internal control so that we can eliminate the possibility of false positives or negative
True
(T/F)
In multiplex PCR, the amplicon sizes of your different primer sets should be DIFFERENT ENOUGH in terms of the number of base pairs / molecular weight so that they can be visualized properly using gel electrophoresis.
True
Although Multiplex PCR can reduce costs and time to simultaneously detect several pathogens in a specimen, it is often _______ than single-primer-set PCR
less sensitive
(T/F)
Multiplex PCR can only be designed for a single-template PCR reaction containing several sets of primers
False.
Multiplex PCR can be designed in either single-template or multiple-template PCR reaction,
Multiplex PCR is more complicated due to the possible interaction of different primers that has _____-_____ possibilities
primer-dimer
This PCR can save costly polymerase and template in short supply.
Multiplex PCR
This PCR increases the accuracy of the molecular PCR because it transforms the exponential data to digital signals
Digital PCR
(T/F)
Digital PCR can be applied if we want to detect and quantify low levels of pathogen sequences
True
This PCR allows for quantification of individual DNA molecules
Digital PCR
The Digital PCR computation is dependent on the ______ distribution
Poisson distribution
(T/F)
Digital PCR can be applied if we want to detect and quantify the expression of rare genetic sequences in single cells
True
(T/F)
Digital PCR can be applied if we want to detect and quantify the clonal amplification of nucleic acids for sequencing mixed nucleic acid samples.
True
This test is done to make sure if the amplicon is correctly done by way of producing cleavage if a restriction enzyme is used
Restriction enzyme cleavage
Digital PCR is ideal for _______
Cloning
This test is used to check if the base pairs are amplified correctly
Gel electrophoresis
Tests to Identify the Quality of PCR product
- Gel electrophoresis
- Capillary electrophoresis
- Restriction enzyme cleavage
- Nucleic acid hybridization
- DNA sequencing
- Real-time quantitative PCR
Cutter GRNDR
This test has the same principle as gel electrophoresis, however uses a different medium (capillary sieving gel)
Capillary electrophoresis
This test can be combined with a probe or a molecule that detects a specific sequence in the amplicon
Nucleic acid hybridization
What test is this?
An enzyme only reacts to a particular amplicon or gene sequence, if it cleaves = correct PCR reaction performed
Restriction enzyme cleavage
In this test, if an amplification is found = DNA is copied or amplified
Real-time quantitative PCR
This test is performed in a machine to check if the applied DNA base pairs are correct
DNA sequencing
This PCR is more complex because in PCR we use fluorometry for measuring PCR output.
Quantitative PCR / qPCR
In performing conventional PCR, what is usually expected is the amplified DNA or the _______.
So in order to quantify how much DNA is produced, we must use this special type of PCR called _ _ _ _.
amplicon
qPCR
In qPCR, aside from the forward and reverse primer, there is another sequence, which is the _____.
Probe
This DNA sequence is connected to a fluorescent marker, and are expected to fit within the sequences produced by the forward and reverse primers.
Probe
(T/F)
DNA is used to detect the presence of a gene
RNA is used to detect the level of expression of the gene
True
Where and when should we use PCR?:
To know the _______ of a ________
Ex.
To identify the presence of hepatitis B virus, we must look for a gene that is only specific to Hepa B virus.
presence of a gene expression
(T/F)
In order to perform RNA PCR, reverse transcription should be performed to make DNA from RNA.
The product will be the formation of complementary DNA (cDNA) from the RNA molecule.
cDNA is now used for PCR.
True
Top Applications for PCR
- Gene expressoin
- Genotyping
- Cloning
- Mutagenesis
- Methylation Analysos
- Sequencing
- Medical, forensics, and applied sciences
+/+
-This is the normal gene
- Here, both strands of your DNA are normal
Wildtype +/+
In RT-PCR, _______ _______ will bind DNA to your RNA template.
After it reaches your (Answer 1), you now have a ___-___ ______
Summary:
From an RNA molecule, we subjected it to reverse transcription, forming a complementary DNA, and then we can use that for PCR.
reverse transcriptase
DNA-RNA hybrid
+/-
-This means that of the copies do not match the wild type
Heterozygous +/-
This is a method that allows you to look into the differences in the nucleotide bases in each allele
It can detect mismatched nucleotide bases (Ex. when adenine binds with cytosine)
Allelic Discrimination
-/-
-If you have both copies that do not match the wild type
Homozygous -/-
Different types of Modern PCR Method
Direct
Indirect
Future of PCR
- Copying larger pieces of DNA
- Miniaturization of hardware (chip-sized)
- Computer automated test and analysis
- Mobile testing
- Rapid turnaround time
