(M) L1: PCR: Pre-Analytic and Optimization Flashcards
1
Q
An in-vitro enzymatic reaction to amplify a defined DNA region that was developed during 1985
A
Polymerase Chain Reaction (PCR)
2
Q
T or F
PCR is a vito enzymatic reaction
A
F (in-vitro enzymatic reaction)
3
Q
One of the most useful techniques where DNA concentration increases exponentially at each cycle
A
Polymerase Chain Reaction (PCR)
4
Q
T or F
In every PCR cycle, the amplification products become template for next rounds
A
T
5
Q
Why is PCR one of the most useful techniques?
A
Because of its sensitivity and speed
6
Q
Prior PCR, old methods took how long to amplify DNA?
A
Days to Weeks
7
Q
PCR amplifies DNA for how long?
A
1-3 hours only wow
8
Q
T or F
PCR require large amounts of DNA
A
F (PCR require small amounts of DNA)
9
Q
What fields is PCR useful for?
A
Research, Genetics, Forensics
10
Q
This encompasses all the procedures before specimen are measured by the analyzer
A
Pre-analytical phase
11
Q
Give me at least three examples of pre-analytical phase (or you can use this for familiarization idk up to you)
A
test selection
patient identification
collection and handling of the sample
sorting
pipetting
centrifugation
disinfection
optimization of reagents, etc
12
Q
T or F
Negligence in pre-analytical phase of PCR does not change the results
A
F (leads to erroneous results)
13
Q
Is the number one enemy in the laboratory which can originate from any point in the procedure
A
Contamination
14
Q
This is the major concern in contamination
A
Laboratory design
15
Q
T or F
In the laboratory, when contamination occur, contaminants can also be amplified by PCR
A
T so much (even a single molecule can give rise to product)
16
Q
What to implement to prevent contamination?
A
Contamination Control
17
Q
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)
A
Amplicon aerosol
18
Q
Potential contamination sources in pre-analytical phase: amplicon aerosol
Can prevent amplicon aerosol
A
real-time PCR (since you don’t need to open tubes prior gel electrophoresis)
19
Q
Potential contamination sources in pre-analytical phase: amplicon aerosol
Cross contamination can cause what results?
A
False-positive for initially negative samples
20
Q
Identify what potential contamination sources in pre-analytical phase:
The actual DNA segment to be amplified is contaminated prior to the procedure
A
Target template contaminants
21
Q
Potential contamination sources in pre-analytical phase: target template contaminants
What 2 procedures can cause target template contaminant?
A
- improper wearing of PPE
- Improper extraction of sample
22
Q
Potential contamination sources in pre-analytical phase: target template contaminants
If PPE are worn incorrectly, what could happen to the final product?
A
Nucleic acids are incorporated
Becauseof the DNA from out body (hair, saliva etc.) nakaklabas
23
Q
Identify what potential contamination sources in pre-analytical phase:
This involves contamination of reagents which is acquired by forgetting to cover reagents, etc.
A
Reagent contamination
24
Q
What must be the pressure of the reagent preparations rooms?
A
Positive (outward; para walang contaminants makapasok)
25
Potential contamination sources in pre-analytical phase: reagent contamination
Once the reagent is contaminated what should you do?
Throw out entire container
26
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
27
Potential contamination sources in pre-analytical phase: reagent contamination
Reagent contamination lead to what results?
Error (Multiple bands show up)
28
What are the 3 Potential contamination sources?
1. Amplicon aerosol
2. Target template contaminants
3. Reagent contamination
29
What are the 7 ways to control contamination?
1. Space and Time preparation
2. Equipment in PCR Laboratory
3. Pre-PCR and Post-PCR setup
4. DUTP-UNG system
5. Environmental conditions
6. Sterilization of reagents
7. Contamination control
30
Identify ways to control contamination:
There should be enough space and time for the procedure
Space and Time preparation
31
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
32
T or F
It is recommended that there should only be one room for every laboratory procedure
F (recommended to have separate rooms)
33
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
34
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
35
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
36
T or F
In crime scenes, gloves are wiped with bleach and air dried before handling the evidence
T
37
7 ways to control contamination: equipment in PCR laboratory
Pipettes, changing tips, isolation cabinets BSCs are subjected under what?
UVL decontamination treatment
38
T or F
The efficiency of UVL decontamination treatment depends on wavelength, energy, and distance
T
39
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
40
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
41
7 ways to control contamination: pre-PCR and post-PCR setup
Clean area of the pre-PCR procedures should be further divided into what?
1. Sample processing rooms
2. Master mix preparation rooms
42
7 ways to control contamination: pre-PCR and post-PCR setup
What 2 equipments are used for master mix preparation?
Box cabinet, BSCs
43
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?
1. Amplification room
2. Visualization room
44
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)
45
T or F
materials such as gloves should be present in pre-PCR only
F (pre and post PCR)
46
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
47
7 ways to control contamination: DUTP-UNG system
What is the full meaning of DUTP-UNG system?
“Deoxyuridine Triphosphate” and “Uracil DNA Glycosylase”
48
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
49
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
50
7 ways to control contamination: DUTP-UNG system
What is the incubation period of UNG enzyme?
50ºC for 2-10 minutes
51
T or F
A sign that UNG enzyme is degraded is once renaturation occurs
F (once denaturation occurs)
52
Identify ways to control contamination:
Positive and Negative pressured rooms in lab
Environmental conditions
53
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)
54
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
55
T or F
Negative pressure rooms are clean areas
F (are dirty areas because of amplicons)
56
7 ways to control contamination: Environmental conditions
Give me an example where positive pressure is used
Reagent preparation
57
7 ways to control contamination: Environmental conditions
Give me an example where negative pressure is used
DNA and RNA isolation, amplification, template-adding
58
T or F
If there is a BSC present, there is no need for pressurization except for when the sample is COVID
T
59
Identify ways to control contamination:
Autoclaving all materials to be used (e.g. jars, pipette tips, etc.)
Sterilization of reagents
60
Identify ways to control contamination:
Positive and negative controls to know and ensure accuracy of assay
Contamination control
61
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
62
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
63
T or F
Negative controls can manifest/show result
F (it only checks if sample is contaminated)
64
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
65
If you see this card, review the Laboratory Designs acc. to Increasing Size
this important so go now, read it
66
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
67
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)
68
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
69
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
70
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
71
T or F
Rigorous hand movements may cause aerosols and contaminations, so it is important to minimize it
T so much
72
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)
73
Placing reagents on ice prevents what?
Nuclease activity (that may breakdown nucleic acid functionality)
74
→ Best done in closed systems
→ Protects the sample against contamination
→ Quality assessment of nucleic acid
Specimen Preparation
75
Specimen Preparation:
In blood and bone marrow specimen what must be inhibited?
Clotting
76
Specimen Preparation:
What anticoagulants should be used to inhibit clotting and chelates Magnesium and Calcium in sample
EDTA and Citrate
| chelate or bind
77
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)
78
Specimen Preparation:
This anticoagulant is not recommended because it inhibits PCR functions
Heparin
78
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.
78
Identify what factor under Specimen Preparation:
Establish the minimum acceptable yield and purity (the limits indicate good amplification)
Quality assessment of nucleic acid
79
Specimen preparation: quality assessment of nucleic acid
What equipment is used to know amount of nucleic acid extracted?
Spectrophotometer
80
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
81
T or F
Consider the stability of nucleic acid and reagents
T so much
82
This is the process of making something fully perfect, functional, or effective as possible
PCR Optimization
83
This is the optimization of reaction conditions is done in order to obtain successful results
ex: right concentrations, temperatures, etc.
PCR Optimization
84
This is the optimizations of various techniques vary across each one
PCR Optimization
85
T or F
In PCR optimization, proper adjustment must be done accordingly
T
86
What are the 4 factors affecting PCR?
1. Primer design
2. Reaction buffer and additives
3. Denaturation, annealing, and elongation temperature and time
4. Cycle number
87
Understanding reagent functions is critical when deciding how to best alter the reaction conditions to obtain the desired product
Manipulating PCR Reagents
88
T or F
Wrong concentrations lead to false results
T
89
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)
90
Success of PCR may rely on changing the concentration of what reagents?
MgCl2, dNTPs, primers, template DNA, or DNA polymerase
91
Are critical components of the reaction which determines the specificity of the PCR
Primers
92
Are short single-stranded DNA fragments that anneal to the upstream and downstream regions of the DNA to be amplified
Primers
93
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)
94
T or F
Primers can only be readily bought
F (You can also design your own primer)
95
T or F
Designing your primer leads to more specificity depending on the nature of the experiment
T
96
T or F
Primer needs to be specific to the target region
T
97
Identify if Do's or Dont's in Principles of Personal Primer Design:
Primer should be 20-30 nucleotides in length
Do's
98
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
99
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
100
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
101
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
102
Do's or Dont's in Principles of Personal Primer Design:
Optimum melting temperature of primers range from?
52-58ºC
103
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
104
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)
105
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)
106
Do's or Dont's in Principles of Personal Primer Design:
Optimal G≡C content should range between?
40-60%
107
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
108
Identify if Do's or Dont's in Principles of Personal Primer Design:
Secondary structures (hairpins) within each primer
Don'ts
109
Do's or Dont's in Principles of Personal Primer Design:
T or F
Hairpin loops cause potential dimerization
T
110
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
111
Identify if Do's or Dont's in Principles of Personal Primer Design:
Dinucleotide repeats/annealing withself
Dont's
112
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
113
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
114
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
115
Increases primer concentration leads to what?
| or TOO HIGH PRIMER CONC.
## Footnote
s
Non-specific products and primer dimers
116
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
117
# Magnesium Concentration:
Optimal concentration for Taq DNA Polymerase
1.5 - 2.0 mM
118
# Magnesium Concentration: (TOF)
(TOF). The final concentration of Mg2+ is
0.05 up to 0.5 mM
False kween (0.5-5.0 mM)
119
# Magnesium Concentration: (TOF)
(TOF). The optimal concentration depends on template, buffer, DNA, and dNTPs.
T
120
Required co-factor for thermostable DNA polymerase and important for successful amplification
Magnesium
121
# Magnesium Concentration:
(TOF) DNA polymerase will stop extending the primer without [Mg2+]
F (can still extend but not that active w/out Mg2+)
122
# 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
## Footnote
(remember that w/out or less Mg2+, tamad c DNA polymerase hence the reduced products)
123
# Magnesium Concentration: (TOF)
Too much or higher concentration of [Mg2+] yields better PCR products
F (higher [Mg2+] results in undesired products)
## Footnote
nag ↑ sensitivity = continuous amplification → decreased **specificity**
124
Typical concentration of each piece of Deoxynucleotides (dNTPs)
200 µM
125
Building blocks of new DNA strands
dNTPs
126
# Deoxynucleotides (dNTPs): TOF
Lower concentrations of dNTPs (50-100 µM)
enhance both the specificity and fidelity of the
reaction, but reduces yield
T
127
# Deoxynucleotides (dNTPs): TOF
Higher concentrations of dNTPs increase
yields particularly in long PCR but can reduce
fidelity and can inhibit PCR
T tlga
128
First invented/discovered/performed PCR by using DNA polymerase isolated from e.coli
Kary Mullis
129
What do you call the enzyme derived from a thermophilic bacterium
Taq Polymerase
## Footnote
Thermus (genus); Aquaticus (species) [aq ay deadicus na]
130
# 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!!)
## Footnote
dna poly can now resist higher temp hehe
131
# Thermostable DNA polymerase:
👹Enumerate👹 other enzymes aside from Taq
Thermus thermophilus
(Tth) and Pfu
## Footnote
pfu not mentioned in lab lec but nasa trans so okay?
132
# Thermostable DNA polymerase: TOF
If the concentration of the DNA polymerase is
low = less, insufficient, or no products
T
## Footnote
ket marami iyong dNTPs, kung less ang dna poly insufficient ang ma-elongate ng primer
133
# Thermostable DNA polymerase: TOF
Too much DNA polymerase will increase the SPECIFICITY
F (decrease)
## Footnote
too much polymerase increases the synthesis of nonspecific pcr products
134
# 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...
## Footnote
... with difficult templates
135
# 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
136
# TOF
The template DNA could be complementary DNA, genomic DNA, or plasmid DNA
T
137
# TOF
Optimal template amount varies based on the type of DNA polymerase used
T
138
# 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
139
# Template DNA
Too much template over the primer would result to what?
Mispriming and may also inhibit the polymerase
140
# Template DNA
Normally, the target sequences to be amplified should be kept at how many kilobases
< 3 kb
## Footnote
LESS THAN HINDI PUSO
141
# Template DNA: (TOF)
Fragments up to 10 kilobases cannot be amplified
F (pwede but longer time to amplify)
142
# Template DNA: (TOF)
You should always check the quality and purity of your DNA templates before amplification
T, yes naman be competent, dear RMT
143
PCRs are carried out in a ________ that will provide a suitable chemical environment for the DNA polymerase
Buffer babe, buffer.
144
# 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
145
Buffer pH usually ranges from
8 - 9.5
146
# 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
147
# 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)
148
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-
## Footnote
95° C on ppt, but can be expanded to 90-96° C
149
# Denaturation Temp and Duration: (TOF)
We must always incubate DNA in longer and higher temps
F (avoid longer or higher temp)
## Footnote
denaturation lang to hindi ka naman magluluto ng nilaga
150
# 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
151
# Denaturation Temp and Duration
What will then happen if the temperature and duration is too low?
incomplete denaturation and low amplification
152
# Denaturation Temp and Duration:
During **thermocycling** what is the typical temp and duration for denaturation?
15-30 seconds denaturation at 95°C
153
# 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
154
# 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
155
When you see multiple bands/shadows in electrophoresis what will you adjust to merge the banding?
Annealing temperature
156
# 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
157
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**
158
# Annealing temperature and Duration: (TOF)
Annealing temperature determines the hybridization between the primer and polymerase
F (primer and template)
159
# Annealing Temperature and Duration
What should you check before setting the annealing temperature?
Melting Temperature
160
# 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)
161
# 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
## Footnote
b (real)
SIKE YOU C. COMPUTE
| HERE FORMULA: Tm = [4 x (G+C)] + [2 x (A + T)] °C
162
# Annealing Temp and Duration:
This card be reminder that thou shouldst aral the given formula for melting temp
amen
163
# Annealing Temperature and Duration: (TOF)
More G-C content on the primer = lower melting temp
F (higher melting temp)
164
# Annealing Temp and Duration: (TOF)
If annealing temp is too low, then it could lead to nonspecific amplification
T
165
# 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
166
# Annealing temp and duration:
What is the typical duration for annealing?
15-30 seconds
167
# Extension time
Generally, how long would be the extension time per 1000 base pairs?
One minute
168
# Extension time
what would be the extension time for a 5 kb product?
5 minutes
| 1 kb = 1000 bp
169
# Extension time:
For products less than 1 kb, how long is the extension time?
45-60 seconds
170
# Extension time: (TOF)
Longer extension time will increase specificity
and number of cycles, however, will give
nonspecific products
F (sensitivity)
171
# Extension time:
If you've set your cycles lower/fewer what does that say about your template concentration?
template concentration = high
172
