(M) L1: PCR: Pre-Analytic and Optimization Flashcards
An in-vitro enzymatic reaction to amplify a defined DNA region that was developed during 1985
Polymerase Chain Reaction (PCR)
T or F
PCR is a vito enzymatic reaction
F (in-vitro enzymatic reaction)
One of the most useful techniques where DNA concentration increases exponentially at each cycle
Polymerase Chain Reaction (PCR)
T or F
In every PCR cycle, the amplification products become template for next rounds
T
Why is PCR one of the most useful techniques?
Because of its sensitivity and speed
Prior PCR, old methods took how long to amplify DNA?
Days to Weeks
PCR amplifies DNA for how long?
1-3 hours only wow
T or F
PCR require large amounts of DNA
F (PCR require small amounts of DNA)
What fields is PCR useful for?
Research, Genetics, Forensics
This encompasses all the procedures before specimen are measured by the analyzer
Pre-analytical phase
Give me at least three examples of pre-analytical phase (or you can use this for familiarization idk up to you)
test selection
patient identification
collection and handling of the sample
sorting
pipetting
centrifugation
disinfection
optimization of reagents, etc
T or F
Negligence in pre-analytical phase of PCR does not change the results
F (leads to erroneous results)
Is the number one enemy in the laboratory which can originate from any point in the procedure
Contamination
This is the major concern in contamination
Laboratory design
T or F
In the laboratory, when contamination occur, contaminants can also be amplified by PCR
T so much (even a single molecule can give rise to product)
What to implement to prevent contamination?
Contamination Control
Identify what potential contamination sources in pre-analytical phase:
PCR products from previous amplifications when opening PCR tubes for gel electrophoresis wherein liquid may spread through air and contaminate other tubes (cross-contamination)
Amplicon aerosol
Potential contamination sources in pre-analytical phase: amplicon aerosol
Can prevent amplicon aerosol
real-time PCR (since you don’t need to open tubes prior gel electrophoresis)
Potential contamination sources in pre-analytical phase: amplicon aerosol
Cross contamination can cause what results?
False-positive for initially negative samples
Identify what potential contamination sources in pre-analytical phase:
The actual DNA segment to be amplified is contaminated prior to the procedure
Target template contaminants
Potential contamination sources in pre-analytical phase: target template contaminants
What 2 procedures can cause target template contaminant?
- improper wearing of PPE
- Improper extraction of sample
Potential contamination sources in pre-analytical phase: target template contaminants
If PPE are worn incorrectly, what could happen to the final product?
Nucleic acids are incorporated
Becauseof the DNA from out body (hair, saliva etc.) nakaklabas
Identify what potential contamination sources in pre-analytical phase:
This involves contamination of reagents which is acquired by forgetting to cover reagents, etc.
Reagent contamination
What must be the pressure of the reagent preparations rooms?
Positive (outward; para walang contaminants makapasok)
Potential contamination sources in pre-analytical phase: reagent contamination
Once the reagent is contaminated what should you do?
Throw out entire container
Potential contamination sources in pre-analytical phase: reagent contamination
What should you do in opening containers?
Write the date when it was opened so next user has an idea
Potential contamination sources in pre-analytical phase: reagent contamination
Reagent contamination lead to what results?
Error (Multiple bands show up)
What are the 3 Potential contamination sources?
- Amplicon aerosol
- Target template contaminants
- Reagent contamination
What are the 7 ways to control contamination?
- Space and Time preparation
- Equipment in PCR Laboratory
- Pre-PCR and Post-PCR setup
- DUTP-UNG system
- Environmental conditions
- Sterilization of reagents
- Contamination control
Identify ways to control contamination:
There should be enough space and time for the procedure
Space and Time preparation
7 ways to control contamination: space and time preparation
What should you do if there is little space in laboratory?
Manage time and distribute tasks through different time periods
T or F
It is recommended that there should only be one room for every laboratory procedure
F (recommended to have separate rooms)
Identify ways to control contamination:
→ There should be gloves dedicated to pre or post procedures
→ Wherever the equipment is designated, it should stay there and not be transferred to other rooms
Equipment in PCR laboratory
7 ways to control contamination: equipment in PCR laboratory
What do you use to wipe bench tops, hoods, or any surface that was in contact with the specimen to degrade the DNA and RNA
10% bleach
7 ways to control contamination: equipment in PCR laboratory
After wiping the bench tops with 10% bleach, what should you do to avoid rust and ensure sample remnants are destroyed
70% ethanol
T or F
In crime scenes, gloves are wiped with bleach and air dried before handling the evidence
T
7 ways to control contamination: equipment in PCR laboratory
Pipettes, changing tips, isolation cabinets BSCs are subjected under what?
UVL decontamination treatment
T or F
The efficiency of UVL decontamination treatment depends on wavelength, energy, and distance
T
7 ways to control contamination: equipment in PCR laboratory
Skin and eye exposure to the UVL can cause?
Mutations and damage to plastics
correction:mutation to cell if for tao; damage to plastics if sa equip
Identify ways to control contamination:
The clean area of the pre-PCR and dirty area of post-PCR procedures should be further divided
Pre-PCR and Post-PCR setup
7 ways to control contamination: pre-PCR and post-PCR setup
Clean area of the pre-PCR procedures should be further divided into what?
- Sample processing rooms
- Master mix preparation rooms
7 ways to control contamination: pre-PCR and post-PCR setup
What 2 equipments are used for master mix preparation?
Box cabinet, BSCs
7 ways to control contamination: pre-PCR and post-PCR setup
The dirty area of the post-PCR procedures should be further divided into what?
- Amplification room
- Visualization room
T or F
Post-PCR does not need to have separate room since it does not have any effect
F (should be separated from other rooms due to the high concentration of DNA)
T or F
materials such as gloves should be present in pre-PCR only
F (pre and post PCR)
Identify ways to control contamination:
A process wherein the enzyme uracil-N glycosylase is added to the reaction/PCR mix at the beginning of each PCR
DUTP-UNG system
7 ways to control contamination: DUTP-UNG system
What is the full meaning of DUTP-UNG system?
“Deoxyuridine Triphosphate” and “Uracil DNA Glycosylase”
7 ways to control contamination: DUTP-UNG system
What enzyme is added to PCR mix at the beginning of each PCR which degrades any nucleic acid containing uracil
uracil-N glycosylase
T or F
uracil-N glycosylase is added to PCR mix to prevent carry-over contamination from previous PCR products so that no further amplification can be done
T
7 ways to control contamination: DUTP-UNG system
What is the incubation period of UNG enzyme?
50ºC for 2-10 minutes
T or F
A sign that UNG enzyme is degraded is once renaturation occurs
F (once denaturation occurs)
Identify ways to control contamination:
Positive and Negative pressured rooms in lab
Environmental conditions
7 ways to control contamination: Environmental conditions
→ This type of pressured room allow for the room to have higher pressure than the surrounding areas hence contaminants cannot enter the room
→ airflow is outward
Positive (kasi if palabas yung toxic na tao sa buhay mo, positive life mo)
7 ways to control contamination: Environmental conditions
→ This type of pressured room decrease the pressure makes the air flow inward hence no contaminants can get out
→ Usually for dirty areas
→ airflow is inward
Negative (kasi if pinapasok mo yung toxic na tao sa buhay mo, negative life mo
T or F
Negative pressure rooms are clean areas
F (are dirty areas because of amplicons)
7 ways to control contamination: Environmental conditions
Give me an example where positive pressure is used
Reagent preparation
7 ways to control contamination: Environmental conditions
Give me an example where negative pressure is used
DNA and RNA isolation, amplification, template-adding
T or F
If there is a BSC present, there is no need for pressurization except for when the sample is COVID
T
Identify ways to control contamination:
Autoclaving all materials to be used (e.g. jars, pipette tips, etc.)
Sterilization of reagents
Identify ways to control contamination:
Positive and negative controls to know and ensure accuracy of assay
Contamination control
7 ways to control contamination: contamination control
Ensures that enzymes are active, buffers are optimal, primers are priming, sequences are correct, and thermocyclers are working
Positive controls
7 ways to control contamination: contamination control
aka “contamination or reagent control”
Ensures that the reaction mix is not contaminated by template DNA or amplified products from previous runs, only the reagent is present
Negative controls
T or F
Negative controls can manifest/show result
F (it only checks if sample is contaminated)
7 ways to control contamination: contamination control
Use DNA sequences that lack the target sequence to ensure that the primers will not anneal to non-target regions (checks primer specificity)
Negative template controls
If you see this card, review the Laboratory Designs acc. to Increasing Size
this important so go now, read it
Identify the PCR Laboratory Setup:
Positive displacement pipettes, Gloves and laboratory coat, Refrigerator, Water bath or Dry heat block, Laminar flow BSC, Cell lysis reagents
Specimen preparation
Identify the PCR Laboratory Setup:
Amplification reagents and supplies, Positive displacements pipettes, Laminar flow BSC, Gloves and Laboratory coat, Water bath or Dry heat block
Reagent preparation (only diff with specimen preparation is wala siyang ref, cell lysis reagents)
Identify the PCR Laboratory Setup:
Thermal cycler, Pipettors, Detection unit (electrophoresis unit, incubator, late washer and reader water bath), Refrigerator and Freezer, Reagents and Supplies for detection
Amplification and Detection
What are the reagents for PCR that needs to be put in freshly filled ice bucket? (or use this as familiarization idk up to you)
Primers, DNA Polymerase, dNTPs, DNA Template, Buffer, Water
How are reagents organized in the workbench? (or use this as familiarization idk up to you)
PCR Tubes, PCR rack, Ethanol-resistant marker, Micropipettors, Thermal cycler, Master mix, Electrophoresis Apparatus
T or F
Rigorous hand movements may cause aerosols and contaminations, so it is important to minimize it
T so much
T or F
if the materials are already close by, there is more movement therefore less airborne particles will be produced
F (there is less movement, kasi nga close na sila)
Placing reagents on ice prevents what?
Nuclease activity (that may breakdown nucleic acid functionality)
→ Best done in closed systems
→ Protects the sample against contamination
→ Quality assessment of nucleic acid
Specimen Preparation
Specimen Preparation:
In blood and bone marrow specimen what must be inhibited?
Clotting
Specimen Preparation:
What anticoagulants should be used to inhibit clotting and chelates Magnesium and Calcium in sample
EDTA and Citrate
chelate or bind
T or F
Magnesium and Calcium are cations that destabilize ribonuclease which help to amplify DNA or RNA, hence need to be activated)
F (stabilize ribonuclease which destroy DNA or RNA, hence need to be deactivated)
Specimen Preparation:
This anticoagulant is not recommended because it inhibits PCR functions
Heparin
Specimen Preparation:
What are the PCR inhibitors? (or use this card for familiarization idk up to you)
Heparin, Hemoglobin, Urea, Excess KCl & NaCl, Other salts, Ionic detergents, Alcohols, etc.
Identify what factor under Specimen Preparation:
Establish the minimum acceptable yield and purity (the limits indicate good amplification)
Quality assessment of nucleic acid
Specimen preparation: quality assessment of nucleic acid
What equipment is used to know amount of nucleic acid extracted?
Spectrophotometer
Specimen preparation: quality assessment of nucleic acid
Deals with the presence or absence of contaminants and whether the extracted sample was pure DNA or RNA
Sample purity
T or F
Consider the stability of nucleic acid and reagents
T so much
This is the process of making something fully perfect, functional, or effective as possible
PCR Optimization
This is the optimization of reaction conditions is done in order to obtain successful results
ex: right concentrations, temperatures, etc.
PCR Optimization
This is the optimizations of various techniques vary across each one
PCR Optimization
T or F
In PCR optimization, proper adjustment must be done accordingly
T
What are the 4 factors affecting PCR?
- Primer design
- Reaction buffer and additives
- Denaturation, annealing, and elongation temperature and time
- Cycle number
Understanding reagent functions is critical when deciding how to best alter the reaction conditions to obtain the desired product
Manipulating PCR Reagents
T or F
Wrong concentrations lead to false results
T
T or F
When troubleshooting the PCR, two reagent should be manipulated at a time
F (only one reagent should be manipulated at a time)
Success of PCR may rely on changing the concentration of what reagents?
MgCl2, dNTPs, primers, template DNA, or DNA polymerase
Are critical components of the reaction which determines the specificity of the PCR
Primers
Are short single-stranded DNA fragments that anneal to the upstream and downstream regions of the DNA to be amplified
Primers
What are the two orders in primers?
forward and reverse primers (one is for the 3’ strand while the other is for the 5’ strand)
T or F
Primers can only be readily bought
F (You can also design your own primer)
T or F
Designing your primer leads to more specificity depending on the nature of the experiment
T
T or F
Primer needs to be specific to the target region
T
Identify if Do’s or Dont’s in Principles of Personal Primer Design:
Primer should be 20-30 nucleotides in length
Do’s
Do’s or Dont’s in Principles of Personal Primer Design:
How many nucleotides should be the length of the primer?
20-30 nucleotides, but 17-30 in other books
Do’s or Dont’s in Principles of Personal Primer Design:
T or F
The longer the primer, the more specific and more efficient at hybridization
F (the longer the primer, the more specific but LESS efficient at hybridization)
kc mas longer mas mabagal
Do’s or Dont’s in Principles of Personal Primer Design:
T or F
Anything less than 20 (or 17) is less/nonspecific but more efficient at hybridization
T
kc mas shorter mas mabilis
Identify if Do’s or Dont’s in Principles of Personal Primer Design:
Optimal melting temperatures (Tm) range from 52-58ºC but can be expanded to 45-65ºC
Do’s
Do’s or Dont’s in Principles of Personal Primer Design:
Optimum melting temperature of primers range from?
52-58ºC
Do’s or Dont’s in Principles of Personal Primer Design:
Optimum melting temperature of primers range from 52-58ºC, but can be expanded to what temperature range?
45-65ºC
Do’s or Dont’s in Principles of Personal Primer Design:
T or F
Optimal melting temperatures are dependent on A=T bonds
F (G≡C bonds, as they have more bonds)
Do’s or Dont’s in Principles of Personal Primer Design:
Stronger bonds indicate what in melting temperature?
indicate increased melting temperature (stronger bonds such as G=C)
Do’s or Dont’s in Principles of Personal Primer Design:
Optimal G≡C content should range between?
40-60%
Do’s or Dont’s in Principles of Personal Primer Design:
3’ ends should contain what in order to clamp the primers and prevent “breathing” of ends therefore increasing efficiency
G or C
Identify if Do’s or Dont’s in Principles of Personal Primer Design:
Secondary structures (hairpins) within each primer
Don’ts
Do’s or Dont’s in Principles of Personal Primer Design:
T or F
Hairpin loops cause potential dimerization
T
Do’s or Dont’s in Principles of Personal Primer Design:
These prevent annealing to the target DNA and will instead anneal to itself
Hairpin loops
Identify if Do’s or Dont’s in Principles of Personal Primer Design:
Dinucleotide repeats/annealing withself
Dont’s
Do’s or Dont’s in Principles of Personal Primer Design:
These cause slipping along the primed segment or hairpin loops
Dinucleotide repeats/annealing withself
Do’s or Dont’s in Principles of Personal Primer Design:
Can cause both primers to bind with each other instead of the target DNA (primer dimer)
Single base runs
Do’s or Dont’s in Principles of Personal Primer Design:
Consists of two primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers.
Primer dimer, btw google meaning lang to
Increases primer concentration leads to what?
or TOO HIGH PRIMER CONC.
s
Non-specific products and primer dimers
What primer concentration are ideal based on the photo that ma’am showed?
0.1 or 0.25 because there are no shadow bands present
Magnesium Concentration:
Optimal concentration for Taq DNA Polymerase
1.5 - 2.0 mM
Magnesium Concentration: (TOF)
(TOF). The final concentration of Mg2+ is
0.05 up to 0.5 mM
False kween (0.5-5.0 mM)
Magnesium Concentration: (TOF)
(TOF). The optimal concentration depends on template, buffer, DNA, and dNTPs.
T
Required co-factor for thermostable DNA polymerase and important for successful amplification
Magnesium
Magnesium Concentration:
(TOF) DNA polymerase will stop extending the primer without [Mg2+]
F (can still extend but not that active w/out Mg2+)
Magnesium Concentration: (TOF)
Why would there be no/less PCR products seen if the [Mg2+] concentration is low?
kc bc due to the reduced polymerase activity
(remember that w/out or less Mg2+, tamad c DNA polymerase hence the reduced products)
Magnesium Concentration: (TOF)
Too much or higher concentration of [Mg2+] yields better PCR products
F (higher [Mg2+] results in undesired products)
nag ↑ sensitivity = continuous amplification → decreased specificity
Typical concentration of each piece of Deoxynucleotides (dNTPs)
200 µM
Building blocks of new DNA strands
dNTPs
Deoxynucleotides (dNTPs): TOF
Lower concentrations of dNTPs (50-100 µM)
enhance both the specificity and fidelity of the
reaction, but reduces yield
T
Deoxynucleotides (dNTPs): TOF
Higher concentrations of dNTPs increase
yields particularly in long PCR but can reduce
fidelity and can inhibit PCR
T tlga
First invented/discovered/performed PCR by using DNA polymerase isolated from e.coli
Kary Mullis
What do you call the enzyme derived from a thermophilic bacterium
Taq Polymerase
Thermus (genus); Aquaticus (species) [aq ay deadicus na]
Thermostable DNA polymerase: (TOF)
With the taq polymerase, DNA pol can now be added once in the beginning of the procedure but will degrade after the heating and cooling cycle
FALSE, HINDI! (MAS STABLE NA NGA DAPAT SO MAINTAINED ACTIVITY THROUGHOUT THE PROCEDURE!!)
dna poly can now resist higher temp hehe
Thermostable DNA polymerase:
👹Enumerate👹 other enzymes aside from Taq
Thermus thermophilus
(Tth) and Pfu
pfu not mentioned in lab lec but nasa trans so okay?
Thermostable DNA polymerase: TOF
If the concentration of the DNA polymerase is
low = less, insufficient, or no products
T
ket marami iyong dNTPs, kung less ang dna poly insufficient ang ma-elongate ng primer
Thermostable DNA polymerase: TOF
Too much DNA polymerase will increase the SPECIFICITY
F (decrease)
too much polymerase increases the synthesis of nonspecific pcr products
Thermostable DNA polymerase:
In a typical 50 µL reaction, what is the ideal unit of DNA polymerase to amplify your target DNA?
1 - 2 units
HOWEVER, it may be necessary to adjust the enzyme amt…
… with difficult templates
Thermostable DNA polymerase
In an experiment with inhibitors (heparin, NaCl, etc.) present in the DNA sample, what would be your course of action to optimize PCR yield?
Increase the amount of DNA polymerase
HOWEVER,nonspecific products may appear with higher enzyme concentration
TOF
The template DNA could be complementary DNA, genomic DNA, or plasmid DNA
T
TOF
Optimal template amount varies based on the type of DNA polymerase used
T
Template DNA: (TOF)
With regards to the primer ratio, primers would be able to find their complimentary sequence even if there is too little template
F
Template DNA
Too much template over the primer would result to what?
Mispriming and may also inhibit the polymerase
Template DNA
Normally, the target sequences to be amplified should be kept at how many kilobases
< 3 kb
LESS THAN HINDI PUSO
Template DNA: (TOF)
Fragments up to 10 kilobases cannot be amplified
F (pwede but longer time to amplify)
Template DNA: (TOF)
You should always check the quality and purity of your DNA templates before amplification
T, yes naman be competent, dear RMT
PCRs are carried out in a ________ that will provide a suitable chemical environment for the DNA polymerase
Buffer babe, buffer.
Buffer: (TOF)
It is important to follow buffer recommendations by the enzyme’s supplier, since the optimal PCR buffer is dependent upon the DNA polymerase used.
chrew
Buffer pH usually ranges from
8 - 9.5
Buffer
Why is there a need to add chemical additives or co-solvents in the buffer
- It helps improve the amplification specificity by reducing the mispriming
- Enhance amplification efficiency by removing secondary structures
Buffers (TOF)
Chemical additives or co-solvents above their optimum concentration does not have an effect on PCR
F (it can inhibit the PCR, above nga ng optimum eh)
Recommended initial denaturation temp and duration prior to PCR cycling to fully denature the DNA
90 - 96°C for 2 minutes
*varies depending on source but yan ang ifollow-
95° C on ppt, but can be expanded to 90-96° C
Denaturation Temp and Duration: (TOF)
We must always incubate DNA in longer and higher temps
F (avoid longer or higher temp)
denaturation lang to hindi ka naman magluluto ng nilaga
Denaturation Temp and Duration:
What will happen if the temperature and duration is too high/long?
increase of sensitivity
*possibility of decreasing taq polymerase acitivity
Denaturation Temp and Duration
What will then happen if the temperature and duration is too low?
incomplete denaturation and low amplification
Denaturation Temp and Duration:
During thermocycling what is the typical temp and duration for denaturation?
15-30 seconds denaturation at 95°C
Annealing Temp and Duration
Typical annealing temperature falls in the range of?
50 - 60° C
that is 5° C BELOW the lowest primer’s Tm
Annealing Temperature and duration: (TOF)
The success of the PCR does not depend on maintaining a high ratio of specific to non-specific annealing of the primer molecules
F
When you see multiple bands/shadows in electrophoresis what will you adjust to merge the banding?
Annealing temperature
Annealing Temperature and Duration
What is the difference between annealing and melting temperature?
Annealing temp - process where primer binds to template
Melting temp - temp at which half of the oligonucleotide molecules are single stranded and half are double standed
Why shouldn’t the Annealing Temperature be equal to the Tm?
annealing temp must be low enough to enable hybridization and high enough to prevent mismatched hybrids from forming
Annealing temperature and Duration: (TOF)
Annealing temperature determines the hybridization between the primer and polymerase
F (primer and template)
Annealing Temperature and Duration
What should you check before setting the annealing temperature?
Melting Temperature
Annealing Temp and Duration:
If the melting temperature is 55°C, what would be the annealing temp?
50° C (basta daw 5°C below ng Melting temp)
Annealing Temperature and Duration:
If the melting temp is not given, what should you do?
a. omiyak
b. sisihin si bongbong
c. magcompute
D. tomalon sa jip
b (real)
SIKE YOU C. COMPUTE
HERE FORMULA: Tm = [4 x (G+C)] + [2 x (A + T)] °C
Annealing Temp and Duration:
This card be reminder that thou shouldst aral the given formula for melting temp
amen
Annealing Temperature and Duration: (TOF)
More G-C content on the primer = lower melting temp
F (higher melting temp)
Annealing Temp and Duration: (TOF)
If annealing temp is too low, then it could lead to nonspecific amplification
T
Annealing temperature and duration:
If annealing temp is too high what would be the result?
no amplification
primers c0nt bind to the template cuz it be denatured
Annealing temp and duration:
What is the typical duration for annealing?
15-30 seconds
Extension time
Generally, how long would be the extension time per 1000 base pairs?
One minute
Extension time
what would be the extension time for a 5 kb product?
5 minutes
1 kb = 1000 bp
Extension time:
For products less than 1 kb, how long is the extension time?
45-60 seconds
Extension time: (TOF)
Longer extension time will increase specificity
and number of cycles, however, will give
nonspecific products
F (sensitivity)
Extension time:
If you’ve set your cycles lower/fewer what does that say about your template concentration?
template concentration = high
