CHEM Molecular Techniques Flashcards
Explain the principle of Qiagen Symphony
- AUTOMATED nucleic acid EXTRACTION
- chaotropic salt (Proteinase K) lyse cells = cellular components are released
- MAGNETIC SILICA BEADS are used to ADSORB NUCLEIC ACIDS
- a magnetic rod transfers beads to a series of reaction vessels; DNA is washed to remove contaminants
- DNA eluted by a low salt buffer
Concentration of dsDNA when A(260) = 1.000
50 µg/mL
Concentration of ssDNA when A(260) = 1.000
33 µg/mL
Concentration of ssRNA when A(260) = 1.000
40 µg/mL
If a solution of dsDNA has an A(260) of 0.81, what is the concentration of dsDNA ?
(50 µg/mL / 1.000) = ( x / 0.810), so x = 40.5 µg/mL
List the reagents used in endpoint PCR
- DNA polymerase
- dNTPs
- PCR buffer
- Primers
- MgCl2
NOTE: Master Mixes can include all reagents to reduce repetitive measurements
What is the function of Taq polymerase ?
- used in endpoint PCR
- replicates template DNA
- recognizes 3’ end of primer
What can occur if [dNTPs] is not optimal ?
Taq polymerase can be inhibited
What is the function of PCR buffers ?
- maintains optimal pH = 8.3 for Taq polymerase
- provides cofactors (Mg2+) for enzymatic activity
- provides salt for annealing/ hybridization
What can occur if [salt] is too high in endpoint PCR ?
Taq polymerase can be inhibited
Which formula is used to calculate the reagent amounts required for endpoint PCR ?
C1V1 = C2V2
What is endpoint-PCR ? ID the 3 thermocycles
Agarose gel electrophoresis used to amplify nucleic acids:
1. Denaturation (95°C)
2. Annealing/ hybridization (60°C)
3. Elongation (65°C)
Describe the unidirectional workflow requirements of a molecular lab
- amplicons produced in the post-room cannot re-enter the pre-room
- this also applies to PPE and equipment
How many primers are necessary for endpoint PCR ?
- two primers that are complementary to opposite strands of DNA target sequence
- must have similar melting temperature (Tm)
- provides 3’ end for Taq polymerase
What is the function of Mg2+ in endpoint PCR ?
A required cofactor for Taq polymerase
NOTE: but if [Mg2+] is too high, DNA replication can be inhibited
Why is a master mix used in endpoint PCR ?
Reduces error introduced by repeated pipetting of small volumes
How is the master mix prepared in the “clean room” ? What is added outside of the clean room ?
- all reagents EXCEPT the template is added into a single vessel in the clean room = master mix
- DNA template is added outside the clean room
Denaturation step in the PCR thermocyler program
At 92 - 95°C:
- dsDNA breaks to form ssDNA
- 10 to 30 sec, but GC-rich targets can take longer
- hot start Taq DNA polymerase prevents non-specific DNA replication at lower temp. until desired denaturation temp. is reached
Annealing step in PCR thermocyler program
At 50 to 60°C:
- primers bind to complementary sequences on ssDNA template
- takes ~30 sec
- specific temp is determined by melting point (Tm) of primer and ionic concentration of rxn = (Tm - 5°C)
Tm = 4(G+C) + 2(A+T)
How is Tm of PCR primers calculated ?
Tm = 4(G+C) + 2(A+T)
Extension step in PCR thermocyler program
At 72°C:
- template DNA is doubled
- DNA polymerase adds free dNTDs to 3’-end of annealed primer = forms dsDNA
a). one min/ 1kb
b). 30 sec for targets < 1kb
What is RT-PCR ?
- reverse transcription PCR uses an RNA template instead of DNA
- RNA is converted to cDNA (complementary DNA) via reverse transcriptase = first strand synthesis
What is first strand synthesis ?
When RNA is converted to complementary DNA (cDNA) via reverse transcriptase in RT-PCR
Can mRNA be used as the template in RT-PCR ?
yes, RNA sub-fractions like mRNA can be used instead of total RNA
What is reverse transcriptase ?
- an RNA-dependant DNA polymerase used in RT-PCR
NOTE: normally isolated from Avian Myeloblastosis Virus (AMV) or Maloney Murine Leukemia Virus (MMLV)*
*MMLV is better for RT-PCR bc it has lower endogenous RNAse activity
What do RT buffers contain ?
Reverse transcription buffers:
- REDUCING AGENTS to inhibit RNA template from forming secondary structures with itself
- cofactors for enzyme activity (Mg2+)
- salts that aid in hybridization of primer to template cDNA
Differentiate one-step vs two-step RT-PCR
One-step:
- cDNA and PCR products are synthesized in the same reaction vessel
- uses target (gene)-specific primers
- faster bc less pipetting steps
- lower possibility of contamination bc reaction vessel is never opened
Two-step:
- cDNA is synthesized in one tube and transferred to a second reaction vessel for PCR
- advantageous when there are multiple (gene) targets
- allows storage of cDNA for later use
The size of the pores in agarose __ as the concentration of agarose __
The size of the pores in agarose DECREASES as the concentration of agarose INCREASES
Which is more expensive: TAE or TBE ? Why ?
Tris-borate-EDTA (TBE) is more expensive bc it has a higher buffer capacity
TAE =Tris-acetate-EDTA
Why is loading dye added in DNA gel electrophoresis ?
- adds color to DNA samples to facilitate loading process
- dyes migrate towards anode at predictable rates = monitors sufficient migration
- increases density of DNA/ensures sample sinks down into well
Describe the dye used for visualization in DNA gel electrophoresis
- ETHIDIUM BROMIDE is an intercalating dye
- binds between complementary bp of dsDNA
- fluorochrome is excited by UV light and observed as red light
Why are DNA ladders used ?
- used as a reference to calculate sample molecular weights
- monitors progress of electrophoresis run
- estimates concentration of sample
In DNA gel electrophoresis, DNA samples migrate from __ to __.
In DNA gel electrophoresis, DNA samples migrate from CATHODE (black) to ANODE (red).
Describe DNA capillary gel electrophoresis
- nucleic acid sample is FLUORESCENTALLY-labelled
- uses long silica tube reinforced with polyimide coating
- thin walls efficiently dissipates heat = allows HIGH VOLTAGE = FASTER
- small sample volumes of sample inserted via electrokinetic injection
- DNA migrates in flowable polymer based on mass:charge from cathode TO ANODE
- fluorescent label is excited by a laser = emitted light is detected
Describe Sanger Sequencing
- “DNA termination sequencing”
- random incorporation of fluorescently-labeled ddNTPs
- ddnNTPs lack 3’ hydroxyl (OH) group = DNA replication terminates
- DNA strands of various lengths are formed (each with a terminal ddNTP)
- capillary electrophoresis will detect the emitted light from fluorophores attached to terminal ddNTPs
- each signal is assigned a base code (GCAT) = visualized as an electropherogram
Describe quantitative PCR
- “Real-time PCR”
- NO electrophoresis to detect amplification products
- qPCR products are fluorescently-labeled (SYBR green) = “real-time” detection
- intercalation of SYBR green dyes in dsDNA = signal intensity increases with accumulation of products
Pros and Cons of using SYBR green in qPCR ?
Pros:
- faster testing
- SYBER green is less expensive than target-specific probes
Con: SYBER green lacks specificity; binds non-specifically to dsDNA = false positive signal
Identify 2 most common qPCR probe techniques
- Hydrolysis probes (5’ nuclease)
- Dual hybridization probes
Describe qPCR hydrolysis probes
- “Taqman” is a 5’-nuclease
- probes are complimentary to target
- modified with a 5’-fluorophore and 3’-quencher (complex does not fluoresce)
- during EXTENSION phase of qPCR = Taq DNA polymerase 5’-3’ exonuclease activity CLEAVES PROBE = FLUORESCENT SIGNAL
- detectable fluorescent signal is proportional to amount of PCR product
Describe qPCR dual hybridization probes. When is a signal detected ?
- PCR with fluorescence resonance energy transfer (FRET)
- uses 2 labeled probes that bind PCR product when in close proximity = ENERGY TRANSFER from donor fluorophore to acceptor fluorophore
- during HYBRIDIZATION phase of qPCR = FLUORESCENCE detected = is proportional to PCR product
- FRET uses 2 primers and 2 probes
Analysis of qPCR requires plotting __ against __.
Analysis of qPCR requires plotting FLUORESCENT SIGNAL INTENSITY against CYCLE NUMBER.
T or F: the signal detected during the initial 3-15 cycles of PCR is background noise
TRUE; the signal detected during the initial 3-15 cycles of PCR is background noise
What do values above the threshold represent in a qPCR analysis plot ?
A true amplification product signal
Identify the 4 controls required in a PCR run
- Positive control = POSITIVE
- Negative control = NEGATIVE
- No-template control = NEGATIVE
- Internal positive control = POSITIVE
What does a positive control in PCR do ?
Validates that PCR conditions were sufficient to amplify and detect the target = POSITIVE amplification of target
What does a negative control do ?
Nucleic acid sample does NOT contain target = NEGATIVE for target but amplification of DNA works
What does a no-template control do ?
The NTC contains all reagents EXCEPT for a DNA template = should be NEGATIVE; detection of PCR products indicates contamination
What does an internal positive control do ?
- tests presence of PCR inhibitors
- is simultaneously extracted and amplified with DNA template = POSITIVE
What additional control is required for reverse transcription PCR ?
- RTase is NOT ADDED = NEGATIVE
- RNA cannot be transcribed to cDNA
- presence of PCR product indicates contamination
What A(260/280) is considered “pure” for DNA ?
~1.8
What A(260/280) ratio is considered “pure” for RNA ?
~2.0
What A(260/230) ratio is considered “pure” for both DNA and RNA ?
Approximately 2.0 - 2.2
Why may A(260/280) be low ?
- residual phenol or other reagents from extraction
- protein contamination
T or F: High A(260/280) ratios are indicative of purity problem
FALSE; high A(260/280) ratios are NOT INDICATIVE OF PURITY PROBLEMS
- can indicate issue with spectrophotometer
Why may A(260/280) be high ?
- not associated with contamination; MORESO PROCEDURAL ERRORS
- using an inappropriate blanking solution ie. not similar ionic strength as sample solution
What is a chaotropic agent ? Give an example.
- molecules that distrupt hydrogen bonding between water molecules (intramolecular bonds that fold proteins together) = DENATURANT
Eg. GUANIDINE
What substance is most commonly used in enzymatic digestion (DNA extraction)?
Proteinase K; cleaves adjacent carboxylic groups
