MOLECULAR TECHNIQUES Flashcards
Explain the principle of Qiagen Symphony
- automated nucleic acid purification
- lysis buffer containing chaotropic salt (Proteinase K) lyse cells and cellular components are released
- magnetic silica beads are added; nucleic acids adsorb to silica
- a magnetic rod transfers beads to a series of reaction vessels; DNA is washed to remove contaminants
- DNA eluted in 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 and identify the thermocyler program steps used
- agarose gel electrophoresis used to PCR amplicons
- denaturation, annealing/ hybridization, extension
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
What can occur if [Mg2+] is too high in endpoint PCR ?
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
- no enzymes are added
- DNA template is added outside the clean room
Describe the 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
Describe the annealing step in PCR thermocyler program
At 50 to 60°C:
- 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)
- takes approx. 30 sec
- primers bind to complementary sequences on ssDNA template
How is Tm of PCR primers calculated ?
Tm = 4(G+C) + 2(A+T)
Describe the extension step in PCR thermocyler program
At 72°C (dependent on which polymerase used ie. Taq):
- DNA polymerase adds free deoxynucleotides to 3’-end of annealed primer = forms dsDNA
a). one min/ 1kb
b). 30 sec for targets < 1kb
- template DNA is doubled
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 ?
It is a RNA-dependant DNA polymerase used in RT-PCR
- 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
Is AMV or MMLV better suited for RT-PCR ? Why ?
Moloney Murine Leukemia Virus is better suited bc it has lower endogenous RNAse activity
What do RT buffers contain ?
Reverse transcription buffers contain:
- cofactors for enzyme activity (Mg2+)
- salts that aid in hybridization of primer to template cDNA
- REDUCING AGENTS to inhibit RNA template from forming secondary structures
Differentiate one-step vs two-step RT-PCR
One-step:
- cDNA and PCR 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
If agarose = 0.5%,
Range of separation of linear DNA = ?
1-30 kb
If agarose = 1.0%,
Range of separation of linear DNA = ?
500 bp - 10 kb
If agarose = 1.5%,
Range of separation of linear DNA = ?
200 bp - 3 kb
If agarose = 2.0%,
Range of separation of linear DNA = ?
100 bp - 2.5 kb
Identify the 2 most common DNA electrophoresis buffers
- Tris-acetate-EDTA (TAE)
- Tris-borate-EDTA (TBE)
Which is more expensive: TAE or TBE ? Why ?
TBE is more expensive bc it has a higher buffer capacity
Why is loading dye added in DNA gel electrophoresis ?
- add color to DNA samples for loading process
- dyes migrate towards anode at predictable rates = monitors sufficient migration
- ficoll, glycerol, or sucrose component increases density of DNA sample and ensures sample sinks down/ is not lost in the buffer
Describe the dye used for visualization in DNA gel electrophoresis
- Ethidium bromide is an intercalating dye
- It 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 capillary gel electrophoresis
- used for DNA sequencing and fragment analysis
- nucleic acid sample must be labelled with a fluorescent tag by PCR reaction
- uses long silica tube reinforced with polyimide coating
- thin walls efficiently dissipates heat = allows HIGH VOLTAGE = FASTER
- small volumes of sample are introduced via electrokinetic injection
- DNA migrates through flowable polymer based on mass to charge ratio TOWARDS ANODE
- fluorescent label is excited by a laser = emitted light is detected
Describe Sanger Sequencing
- aka DNA termination sequencing due to random incorporation of fluorescently-labeled ddNTPs
- ddNTPs 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
- aka real-time PCR
- does not require electrophoresis for detecting amplification products
- qPCR products are fluorescently-labeled, allowing for “real-time” detection
- intercalation of SYBR green dyes in dsDNA = signal intensity increases with accumulation of products
Pros and Cons of qPCR, target-specific probes vs SYBER green
Pros:
- better sensitivity
- faster testing
- reduced risk of contamination
- SYBER green is less expensive than target-specific probes
Cons:
- target-specific probes are more expensive
- 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”; 5’-nuclease
- probe is an oligonucleotide sequence 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 the probe = FLUORESCENT SIGNAL
- detectable fluorescent signal is proportional to amount of PCR product
Describe qPCR dual hybridization probes
- PCR with fluorescence resonance energy transfer (FRET)
- uses 2 labeled oligonucleotide probes that bind PCR product in close proximity (5 nucleotides) = ENERGY TRANSFER from donor fluorophore to acceptor fluorophore
- FLUORESCENCE IS DETECTED AT HYBRIDIZATION 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 is the threshold cycle/ crossing point ?
The cycle at which the amplification plot crosses the threshold
What do values above the threshold represent in a qPCR analysis plot ?
A true amplification product signal
The concentration of DNA in a patient sample can be determined by generating a __.
The concentration of DNA in a patient sample can be determined by generating a standard curve.
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 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
Identify 2 types of positive internal controls
- Endogenous; found in specimen ie. actin
- Exogenous; added into samples during extraction or before PCR amplification ie. MS2
What additional control is required for reverse transcription PCR ?
- REVERSE TRANASCRIPTASE is NOT ADDED = NEGATIVE
- RNA cannot be transcribed to cDNA
- presence of PCR product indicates contamination
What is transcription mediated amplification used to detect ?
- Chlamydia trachomatis rRNA
- Neisseria gonorrhoeae rRNA
How is the rRNA in transcription mediated amplification obtained ?
- buffer lyses C. trachomatis and N. gonorrhoeae and protects rRNA
- capture oligo binds complementary rRNA
- poly-adenosine sequence of capture oligo hybridizes to magnetic particle
- magnetic field is applied and captured targets are washed
Describe the 3 functions of reverse transcriptase in transcription mediated amplification (TMA)
- RNA-dependent DNA polymerase = converts RNA to cDNA
- RNAse H activity = digests the RNA of RNA:cDNA hybrid
- DNA dependent DNA polymerase = synthesizes second strand of DNA
When do capture oligo T7 and non T7 bind during transcription mediated amplification (TMA) ?
- T7 binds to target rRNA; provides 3’ for reverse transcriptase TO FORM cDNA
- non T7 binds cDNA; provides 3’ for DNA dependent reverse transcriptase TO FORM dsDNA
How are RNA products in transcription mediated amplification (TMA) detected ? What is this called ?
- RNA products are detected by ssDNA probes labeled with acridinium esters = CHEMILUMINESCENCE
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(260/230) be low ?
- carbohydrate carryover
- residual phenol or guanidine HCl from extraction
- glycogen was used in the precipitation
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 intramolecular bonds that fold proteins together (hydrogen bonding between water molecules) = denaturant
Eg. GUANIDINE
What substance is most commonly used in enzymatic digestion ?
Proteinase K; cleaves adjacent carboxylic groups
