360 - Molecular Techniques Flashcards
The target n________ _____d for detection will determine the starting material for extraction.
nucleic acid
The selection of starting material is dependent on this
the target nucleic acid
potential target nucleic acids
chromosomal DNA, mitochondrial DNA, circulating fetal DNA, and microbial DNA & RNA
The three basic steps in DNA extraction and purification:
cell disruption, separation of DNA from cellular material, and concentration of the DNA
how are DNA cell lysates produced?
DNA cell lysates are produced by disrupting cell walls and membranes
Lysis solutions usually contain one or more of the following reagents:
chaotropic salts – guanidinium isothiocyanate, urea, and sodium dodecyl sulphate (SDS); detergents – SDS; and an alkaline denaturant – sodium hydroxide
NOTE: these agents denature proteins resulting in the disruption of cell membranes and the release of the cellular contents, including DNA
porteinase K can be added
This is also added to lysis buffers to chelate metal ions which are required as cofactors for DNases
EDTA
This can be added to extraction to remove residual RNA
RNase
Processing before lysis: tissue biopsies
They can be disrupted by homogenization using stainless steel beads in a bead mixer or by grinding liquid nitrogen frozen tissue with a mortar or pestle
T or F. Nucleic acids can be extracted from formalin fixed paraffin embedded tissue
T!
Gram-positive bacteria require enzymatic digestion with THIS before cell lysis
lysozyme
Fungal cell walls can be disrupted by enzymes or by using c______ t_________ b_____ in a bead mixer
carbon tungsten beads
how can cell lysates be further purified to separate DNA from other cellular components?
can be cleared by liquid extraction/purification methods like phenol/chloroform or by solid phase isolation like magnetic silica beads
- liquid extraction/purification methods = cellular components are precipitated by centrifugation or removed by filtration
- magnetic bead extraction/purification method = binding of the DNA to the bead surface by adsorptive or ionic interactions
Qiagen Symphony
an automated nucleic acid purification platform
how does the Qiagen Symphony work?
- samples incubated in a lysis buffer containing a chaotropic salt, and usually, Proteinase K
- cells lyse and nucleic acids + other cellular components released
- magnetic silica beads added to lysate, and nucleic acids adsorb to the silica
- magnetic rod transfers the bound nucleic acid material to a series of reaction vessels where the DNA is washed to remove contaminants
- DNA eluted in a low salt buffer.
DNA and nucleotides absorb at which wavelength
260 nm
how do we calculate nucleic acids in a pure solution?
50 μg/mL of double-stranded DNA has an A260 of 1
33 μg/mL of single-stranded DNA has an A260 of 1
40 μg/mL of single-stranded RNA has an A260 of 1
T or F. Both nucleic acid and proteins absorb UV light at 260 and 280 nm.
T!
how can protein contamination of nucleic acid solutions be assessed?
by analyzing the ratio of absorbance at 260:280 nm
pure dsDNA has an A260/A280 ratio of 1.7 to 1.9
pure single-stranded RNA has an A260/A280 ratio of approximately 2.0
T or F. The A260/A280 ratio is affected by alkaline pH
F! acidic pH; slightly alkaline buffer is the preferred solvent
These contaminants can be detected by analyzing the absorbance at 230 nm
carbohydrates, phenol and guanidine
The A260/A230 ratio of pure nucleic acid preparation will be greater than the A260/A280 ratio
what is a Nanodrop?
assess nucleic acid concentration and purity but
used instead of a spec (Nanodrop is faster & more convenient!)
how does a Nanodrop work?
- small volumes of the sample (1-2 μL) are pipetted onto the end of a fibre optic cable
- a second cable is placed on the opposite side of the drop of sample to form a small gap
- a pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear charged couple detect array is used to analyze the light after passing through the sample
- a computer algorithm calculates the concentration of nucleic acid
endpoint or conventional PCR
any PCR protocol which uses agarose gel electrophoresis to visualize the PCR amplicons
This uses fluorescent labels to monitor the production of PCR amplicons as they are synthesized
quantitative PCR aka real-time PCR
PCR is the combination of three processes that occur at different temperatures:
denaturation, annealing/hybridization, and extension
- one round of each = cycle
- standard PCR = 25 to 40 rounds
- number of cycle rounds required is dependent on the amount of template used
- thermal cycler
denaturation
- 92 - 95°C the hydrogen bonds of double-stranded DNA break and single-stranded DNA is formed
- single-stranded DNA serves as the template for a heat resistant DNA polymerase
- 10 to 30 seconds denaturation is sufficient for most targets; GC rich targets can require longer for complete denaturation
This prevents non-specific DNA replication at lower temperatures
hot start Taq DNA polymerase
- enzyme is inactivated by antibodies or chemical modifications until the denaturation temperature is reached
- Hot Start PCR reaction will start with an extended initial denaturation period before cycling
describe annealing
- temperature of the reaction is decreased to around 50 to 60°C for approximately thirty seconds
- temperature is controlled by the melting point (Tm) of the primers and the ionic concentration of the reaction
- starting point for an annealing temperature is the primer Tm - 5°C
- primers bind to their complementary sequences on the single-stranded DNA template
During this step, DNA polymerase to bind the free 3’ end of the newly formed double-stranded DNA and initiates DNA replication
annealing
The Tm of a primer can be calculated using the following formula:
Tm = 4(G + C) + 2(A + T)
DNA polymerase forms double-stranded DNA by adding complementary free deoxynucleotides to the 3’ end of the annealed primer
extension
= DNA has been doubled
The extension temperature of a PCR is dependent on the…
type of DNA polymerase used
Taq polymerase active at 72C
General rule for extension time
1 minute per 1 kilobase and 30 seconds for targets less than 1 kilobase
template DNA
A gene or portion of a gene that serves as the template on which many new copies (amplicons) of the DNA are created
T or F. If you have1 μg of sample genetic DNA it is equivalent to 1 μg of template DNA.
F! It is not
the enzyme that replicates DNA
DAN polymerase
DNA polymerase
- replicates DNA
- each cycle of PCR doubles the copies of the target DNA
- many types
- Taq polymerase is used for endpt PCR
dNTPs
- mixture of the four dNTPs is used to provide the building blocks for the new DNA strands created by PCR
- there is an optimal concentration for dNTPs in the reaction mixture; Otherwise, Taq polymerase can be inhibited.
optimal working pH of Taq
8.3
what do buffers do in PCR
- maintain pH
- provide co-factors for enzyme activity (eg. Mg 2+)
- include salts that aid in the hybridization of primer to template DNA
NOTE: if concentration of salt is too high, polymerase will be inhibited
what are primers?
single-stranded oligonucleotides typically between 18 - 30 nucleotides in length with between 40-60% GC content
the sequence of the primer is _______________ to an upstream or downstream region of the target DNA
complementary
Taq polymerase recognizes the free ___’ end of the bound primer to initiate ______ ___________.
3’ end; DNA replication
T or F. Two primers are necessary, and they must be complementary to opposite strands of a DNA target sequence and have similar melting temperatures (Tm)
T!
these ions are a required cofactor for Taq polymerase activity
Mg 2+
- If the concentration is too high, replication of DNA will be inhibited
- usual concentration for most PCR is 1.5 - 2 mM
purpose of master mixes
- to reduce the error introduced by repeat pipetting of small volumes
- performed in a clean room; all reagents excluding template are mixed
- mixed into a single vessel
- then aliquoted into reaction vessels
- outside clean room = template is added to rxn vessels
- master mixes prepare without enzyme
- stored at -20C
reverse transcription PCR/ RT-PCR
template is RNA instead of DNA
- RNA converted to cDNA by enzyme reverse transcriptase = first strand synthesis
- cDNA used as template for PCR rxn
first strand synthesis process
heat -> hybridize -> reverse transcription -> cDNA
RT-PCR template
- either total RNA or RNA sub-fractions like mRNA can be used as a template
- RNA heated to denature secondary structures (eg hairpins)
RT-PCR primers
can be template specific, random oligomers, or poly dT (for eukaryotic specific mRNA targets)
reverse transcriptase for RT-PCR
- most labs = commercial genetically modified reverse transcriptase: RNA-dependent DNA polymerases
- normally isolated from a variety of retroviral sources such as AMV or MMLV
- MMLV better suited for RT-PCR bc lower endogenous RNase activity
AMV
Avian myeloblastosis virus
MMLV
Moloney murine leukemia virus
dNTPs for RT-PCR
mixture of four dNTPs used to provide building blocks for the new DNA strands created by PCR
RT buffer
- each type of reverse transcriptase has an optimal pH range at which they will react (just like Taq at 8.3)
- often contain reducing agents to inhibit the RNA template from forming secondary structures
One-step vs Two-step RT-PCR
one-step = cDNA and PCR synthesized in same rxn vessel; uses target (gene) specific priers; more time consuming; less likely to be contaminated as rxn vessel is never opened
two-step = cDNA is synthesized in one tube and transferred to a second rxn vessel for PCR; advantageous w their multiple (gene) targets and allows for storage of cDNA for later use
DNA and RNA molecules have a constant charge to mass ratios; thus, the migration rate of nucleic acids is dependent on the…
length of nucleic acid
T or F. The size of the pores in the agarose decrease as the concentration of agarose increases
T! by altering the concentration of agarose the ability to resolve DNA molecules of different lengths can be enhanced
what can happen if the buffering capacity of the electrophoresis buffer is insufficient?
the cathode can become alkaline and the anode acidic
the two most common DNA buffers
Tris-acetate-EDTA (TAE)
Tris-borate-EDTA (TBE); more expensive but higher buffering capacity
These are added to DNA samples before loading the gel (pipetting the DNA into the well)
gel loading buffers
- mixture of dyes and either FFicoll, glycerol, or sucrose
- dye component usually bromophenol blue and xylene cyanol (adds colour to sample and aids the loading process)
T or F. Gel loading dyes migrate towards cathode
F! They migrate towards anode at predictable rates which helps us to monitor electrophoresis to ensure DNA sample of interest has migraed sufficiently for optimum results
Ficoll purpose
increases density of DNA sample to ensure sample sinks in the gel well and is not lost in buffer
Nucleic acids can be visualized in an agarose gel using special dyes. This is the most common dye used
ethidium bromide
- an intercalating dye; it binds in between the complementary base pairs of double-stranded DNA
The fluorochrome of ethidium bromide is typically excited with __ light and is observed as _____ light.
UV; red
commercial alternatives to ethidium bromide (teratogen, mutagen and toxin)
Sybr Green and Gel Red
- also intercalating so also mutagens
Molecular weight markers/standards, also known as DNA ladders, are composed of …
nucleic acid fragments of known sizes and concentrations
what are molecular weight markers used for?
to calculate sample molecular weights
to monitor the progress of an electrophoretic run
to estimate the concentration of the sample
NOTE: a marker should be included in every gel
a technique used for DNA sequencing and fragment analysis
capillary gel electrophoresis
before CGE, the sample nucleic acid must be labelled with …
fluorescent tag by a PCR reaction
the PCR must be cleaned to remove unincorporated label and reagents
explain CGE
- long, thin silica tube with polyimide coating
- internal diameter = 20 to 180 um; with length = 20 to 80 cm
- heat dissipated quickly through thin walls = voltage 100X than gel
- 1-2 μL containing between 1 pg/mL to 1 μg/mL of the labelled template injected into the capillary by electrokinetic injection
- DNA migrate through a ‘flowable polymer’ to the anode based on their mass to charge ratio
- as molecules migrate towards the anode, they pass an exposed area of the capillary = fluorescent label on nucleic acid is excited by a laser, and the emitted light is detected
injection timing in CGE
affects signal intensity and resolution
accurate timing essential for reproducible results
Nucleotides are strung together by _____________bonds which join…
phosphodiester bonds join 3’ hydroxyl group of one dNTP to a 5’ phosphate of an adjoining dNTP
what are ddNTPs?
Dideoxyribonucleotides
- lack 3’ hydroxyl groups and are unable to form phosphodiester bond
- if a ddNTP is incorporated during replication, DNA replication terminates
In dideoxy sequencing, the target DNA is amplified by…
PCR
- mastermix includes both dNTPs and fluorescently labelled ddNTPs
- PCR replication of the target DNA proceeds as normal; however, when a ddNTP is randomly incorporated, replication is terminated
- multiple cycles of amplification = DNA strands of various lengths will be generated, each terminating with a labelled ddNTP
what happens once the amplification program has ended?
- the sequencing reactions are cleaned to remove any unincorporated nucleotides and other reagents
- cleaned sequencing reactions are capillary electrophoresis
- fluorophore attached to the ddNTP will emit a wavelength of light which is detected after pasing fluorescence excitation light source
- computer converts the detector signal into relative fluorescent units and assigns a base code (AGCT) to the signal
- data visualized as an electropherogram
quantitative PCR is also known as..
real-time PCR
quantitative PCR
- real-time
- does not need detection of amplification products by electrophoresis
- qPCR products are fluorescently labelled, allowing ‘real time’ detection of amplification products
- amplicons can be detected by intercalation of fluorescent dyes such as Sybr green OR hybridization of fluorescently labelled probes using an integrated thermal cycler and detector system
advantages to qPCR
- better sensitivity
- faster testing
- reduced risk of contamination
disadvantages of qPCR
cost involved in purchasing specialized equipment & probes as well as the difficulties encountered in optimizing the technique
T or F. Sybr Green is specific when it binds to dsDNA
F! non-specific and it emits a fluorescent signal when excited
how can SyBr green binding in qPCR be detected?
- during the extension steps of the amplification program
- the fluorescent signal intensity increases with the accumulation of amplicon
main advantage of Sybr Green (qPCR)
less expensive than target-specific probes
main disadvantage of Sybr Green (qPCR)
lack of specificity
non-specific PCR products can contribute to the signal
The most common qPCR probe techniques
Hydrolysis and Dual Hybridization probes
NOTE: both probe types are based on FRET
hydrolysis probes
- 5’ nuclease
- an oligonucleotide sequence complementary to the target sequence
- modified with a fluorophore attached to the 5’ end and a quencher attached to the 3’ end
- intact probe does NOT fluoresce
- during extension phase = Taq cleaves the probe separating fluorophore and quencher moiety
- separation of the fluorophore and the quencher results in detectable fluorescence signal that is proportional to the amount of accumulated PCR product
- commercial name Taqman
dual hybridization probes
- PCR with fluorescence resonance energy transfer (FRET) probes uses two labelled oligonucleotide probes that bind to the PCR product in a head-to-tail fashion
- two probes bind = fluorophores in close proximity, five nucleotides = energy transfer from a donor fluorophore to an acceptor fluorophore
- fluorescence is detected during the hybroidization phase of PCR and is proportional to the amount of PCR product
the FRET system uses…
two primers
two probes
Analysis of qPCR requires…
plotting fluorescent signal intensity against cycle number
NOTE: signal detected during the initial 3 -15 cycles of PCR is background noise (baseline of fluorescence; subtracted from fluorescence value obtained from amplification products)
where is threshold value set in qPCR?
set within the linear region of the amplification curve
- any signal above the threshold value = true amplification product signal
- threshold cycle (Ct) or crossing point (Cp) is the cycle at which the amplification plot crosses the threshold
In qPCR, the concentration of DNA in a patient sample can be determined by generating a…
standard curve
How is a standard curve for qPCR generated?
- dilution series of known template concentrations are amplified
- plot template concentrations against their respective crossing points
- standard curve can then be used to determine the initial starting amount of the target template in the patient sample
The CAP requires _______ controls for each qPCR run:
three
positive, negative, and no-template controls
ALSO: assays which are based on the presence or absence of PCR products must include an internal positive control
qPCR: positive control
- contains target nucleic acid of sufficient quantity and purity for amplification
- validates that the PCR conditions were sufficient to amplify and detect the target of interest
qPCR; negative control
- a nucleic acid sample in which the target sequence is not present
- this control validates the specificity of the PCR reaction
qPCR: no-template control
- contains all the reagents necessary for PCR except for the target nucleic acid; water is substituted to make up the volume difference
- detection of PCR products indicates nucleic acid contamination
for PCR to be valid…
positive control pos and both negative and NTC = neg
for PCR to be valid…
positive control pos and both negative and NTC = neg
what is an internal positive control?
used to test for the presence of PCR inhibitors
- the internal control is simultaneously extracted and amplified with the template nucleic acid
- internal positive controls (IC) can be endogenous template normally found in the specimen (e.g. ß-actin, actin, and glyceraldehyde-3-phosphate dehydrogenase)
- exogenous controls are spiked into samples either during nucleic acid extraction or before PCR amplification (e.g. MS2).
PCR rxn with negative internal pos control and negative target
A PCR reaction with both a negative internal positive control and a negative target is suggestive of inhibition. The result of this individual reaction is invalid
RT-PCR control
additional control required for reverse transcription PCR
- set up at transcription step of rxn
- reverse transcriptase enzyme omitted from rxn
- absence of enzyme, RNA cannot be transcribe to DNA = should be NO DNA detected at end of PCR phase of assay
- presence of PCR product = presence of contaminating DNA in RNA template
Transcription mediated amplification (TMA)
utilized for the detection of Chlamydia trachomatis and Neiserria gonorrheae on the Panther instrument
- assay detects rRNA
- prokaryotic organisms lysed in a buffer (also protects rRNA)
- rRNA is bound by a capture oligo
- oligos are complementary to the target rRNA
- capture oligos also have a poly-adenosine sequence which hybridizes to a magnetic particle that has complementary sequence
- after capture of rRNA targets, a magnetic field is applied and the magnetic particles (with and without hybridized rRNA) are immobilized and washed
T7 and TMA process
- oligo (TMA)
- hybridizes to target rRNA
- 5’ end of the T7 oligo encodes the T7 promoter sequence and the 3’ end of the oligo binds the target RNA and serves as a primer for a reverse transcriptase
- after creation of cDNA from the RNA target, the RNase H activity of the reverse transcriptase digests the RNA of the RNA:DNA hybrid molecule
- second oligo, non T7, binds the cDNA and the DNA dependent DNA polymerase activity of the reverse transcriptase synthesis the second strand of DNA
- T7 RNA polymerse binds to the now incorporated T7 promoter and synthesises multiple copies of RNA, identical to the target
- newly synthesised RNA is then amplified by the reverse transcriptase and the T7 and non-T7 oligos
= transcribed by T7 RNA polymerase - RNA products are detected by single stranded DNA probes labeled with acridium esters (chemiluminescence)
three functions of reverse transcriptase in TMA
- RNA-dependent DNA polymerases
- RNaseH activity
- DNA-dependent DNA polymerase activity
