Академический Документы
Профессиональный Документы
Культура Документы
PCR Recipe
Template DNA 50-100ng/l Reaction buffer (Tris-HCl, ammonium ions, KCl), magnesium ions, bovine serum albumin) This buffer provides the ionic strength and buffering capacity needed during the reaction. MgCl2 - 1.5- 3mM dNTPs -Equimolar ratios, 200 M each dNTP Primers (0.1 and 0.5 M) DNA polymerase -1-2 unit/25 l reaction
Colony PCR
Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products. Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 l of TE autoclaved dH2O in an microfuge tube. Heat the mix in a boiling water bath (90-100C) for 2 minutes Spin sample for 2 minutes high speed in centrifuge. Transfer 20 l of the supernatant into a new microfuge tube Take 1-2 l of the supernatant as template in a 25 l PCR standard PCR reaction.
Asymmetric PCR
Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is ideal. PCR is carried out as usual, but with a great excess of one primers for the chosen strand.
Nested PCR
Two pairs (instead of one pair) of PCR primers are used to amplify a fragment. First pair -amplify a fragment similar to a standard PCR. Second pair of primers-nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. Advantage- Very low probability of nonspecific amplification
AFLP PCR
AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA. AFLP can be also used for genotyping individuals for a large number of loci Genomic DNA is digested with one or more restriction enzymes. tetracutter (MseI) and a hexacutter (EcoRI).
Inverse PCR
Inverse PCR (Ochman et al., 1988) uses standard PCR (polymerase chain reaction)- primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been selfligated Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA. Applications Amplification and identification of sequences flanking transposable elements, and the identification of genomic inserts.
Multiplex PCR
Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers.
In Situ PCR
In Situ PCR (ISH) is a polymerase chain reaction that actually takes place inside the cell on a slide. In situ PCR amplification can be performed on fixed tissue or cells. Applies the methodology of hybridization of the nucleic acids. Allows identification of cellular markers Limited to detection of non-genomic material such as RNA, genes or genomes
In Situ PCR
Long PCR
Extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb). Long PCR is useful only if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together. Application- to clone large genes not possible with conventional PCR.
Allows the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.
Allele-specific PCR
Used for identify of SNPs. It requires prior knowledge of a DNA sequence, including differences between alleles. Uses primers whose 3' ends encompass the SNP PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer Successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence
RealTime-PCR
Introduction
Linearity: The intensity of the emitted fluorescent light is a linear function of the amount of fluorochrome present. The signal becomes nonlinear at very high fluorochrome concentrations. Brightness: Fluorochrome differ in intensity. Dull fluorochrome is a less sensitive probe than a bright fluorochrome. The brightness depends on two properties of the fluorochromeIts ability to absorb light (extinction coefficient). The efficiency with which it converts absorbed light into emitted fluorescent light (quantum efficiency). Environmental factors: Environmental conditions can affect the brightness or the wavelength of the absorption or emission peaks.
What is Fluorescence Resonance Energy Transfer (FRET)? FRET is a distance dependent interaction between the excited states of 2 dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon
FRET (contd):
The Donor and Acceptor in close physical proximity (10 -100 Angstrom) can lead to FRET or Quenching
hv hv
A D
(c) No hv
Hybridization probes
hv
hv
R R Q Q
(e) No Physical proximity + hv (Quenching released)
Quantitating Fluorescence
A flurometer exploits the principles of fluorescence to quantitate fluorescent (dye) molecules in the following way:
A strong light source which produces light within a specific light range ( eg xenon arc lamp) is focused down to a tight beam. The tight beam of light is sent through a filter which removes most of the light outside of the target wavelength range. The filtered light beam passes through the liquid target sample striking some of the fluorescent molecules in the sample. Light emitted from the fluorescent molecules travels orthogonal to the excitation light beam pass through a secondary filter that removes most of the light outside of the target wavelength range.
The filtered light then strikes a photodetector or photomultiplier which allows the instrument to give a relative measurement of the intensity of the emitted light.
Instruments
1a. Excitation filters 1b. Emission filters Tungsten halogen light source Microplate format
(350 - 1000nm continuous)
Cycler
dsDNA BindingDye
SYBR Green I SYBR Green II EVA Green LC Green BEBO YO-PRO SYTO family
TaqMan Probe
When intact, the fluorescence of the reporter is quenched due to its proximity to the quencher Probe hybridizes to the target
dsDNA-specific 5'>3' exonuclease activity of Taq or Tth cleaves off the reporter
Reporter is separated from the quencher. Fluorescent signal Signal is proportional to the amount of amplified product in the sample
TaqMan Probe
Advantages
Highly fluorogenic
Easy PCR setup Sequence-specific detection, multiplexing
Disadvantages
Expensive
Probe design and positioning challenging Similar conditions for primers and probes
Loop
Stem
5 3
3 5
Denaturation
5 3 5
Q
5
Operation of Molecular Beacon (MB): MB is non-fluorescent due to close proximity of the nonfluorescent quencher (Q) and the fluorescent Reporter The probe denatures and the loop anneals to the target sequence of the amplicon Separating the quencher from the fluorophore and thereby producing fluorescence which is proportional to the amplicons produced during PCR MB is displaced not destroyed during amplification, because a DNA polymerase lacking 5' exonuclease activity is used
5 3
Extension
5 3 5
5 3
Molecular beacons
Advantages
High specificity, low background
Post PCR analysis PCR multiplex Allelic discrimination (greater specificity than linear probes)
Disadvantages
Challenging design
Long probes less yield Intramolecular competitive binding Low signal levels (proximity of reporter and quencher)
Scorpion Primers
Complementary sequence
5 Reporter
3 Quencher Blocker
PCR primer
The loop of the Scorpions probe includes a sequence that is complementary to an internal portion of the sequence it primes.
During the first amplification cycle, the Scorpions primer is extended, and the sequence complementary to the loop sequence is generated.
After subsequent denaturation and annealing, the loop of the Scorpions probe hybridizes to the internal target sequence, and the reporter is separated from the quencher. The resulting fluorescent signal is proportional to the amount of amplified product in the sample. The Scorpions probe contains a PCR blocker just 3' of the quencher to prevent read-through during the extension of the opposite strand.
The template & probe denature The primer is part of the Scorpion probe
Hybridization Probes
These assays use two sequence-specific oligonucleotide probes in addition to two sequence specific primers. The two probes are designed to bind to adjacent sequences in the target. The probes are labeled with a pair of dyes that can engage in FRET. The donor dye is attached to the 3' end of the first probe, while the acceptor dye is attached to the 5' end of the second probe. During real-time PCR, excitation is performed at a wavelength specific to the donor dye, and the reaction is monitored at the emission wavelength of the acceptor dye. At the annealing step, the probes hybridize to their target sequences in a head-to-tail arrangement. This brings the donor and acceptor dyes into proximity, allowing FRET to occur. The increase in PCR product is proportional to amount of fluorescence
Hybridization probes
Probe 2
Probe 1
FRET
Hybridization probes
h
Probe 2
Probe 1
h D D FAM FRET A A
Amplicon
LC red 640
Hybridization probes
Advantages
Probe with only one fluorophore Easy synthesis and quality controls Reduced background fluorescence High specificity
Disadvantages
Strict compatibility between donor & acceptor fluorophores
FRET D A
PolyA Tail
Sunrise Probe with polyA tail binds to the primer polyT tail at annealing.
Q R
hv
Q
AAAAAAAAAAAAAA
The Sunrise probe changes conformation during denaturation & quenching by DABCYL is removed allowing FITC to fluoresce
Molecular Beacons
19%
FRET probes
15%
9%
9%
Other
3%
Scorpion probes
0%
2%
10%
20%
30%
40%
50%
60%
70%
80%
Detection Chemistries
Initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline of amplification plot. An increase in fluorescence above the baseline indicates detection of accumulated PCR product.
The parameter CT(Threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.
During the exponential phase, none of the reaction components is limiting; as a result, CT values are very reproducible for reactions with the same starting copy number. On the other hand, the amount of PCR product observed at the end of the reaction is very sensitive to slight variations in reaction components.
Threshold cycle CT
The Threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background. The threshold line is set in the exponential phase of the amplification for the most accurate reading. The cycle at which the sample reaches this level is called the Cycle Threshold, CT.
CT value of 40 or more means no amplification and cannot be included in the calculations. A sample whose Ct is 3 cycles earlier than another's has 23 = 8 times more template.
DRn
Rn+ is the Rn value of a reaction containing all components (the sample of interest) Rn- is the Rn value detected in NTC (baseline value)
Standard Curve
A dilution series of known template concentrations can be used to establish a standard curve for determining the initial starting amount of the target template. The log of each known concentration in the dilution series (x-axis) is plotted against the Ct value for that concentration (y-axis). From this standard curve, information about the performance of the reaction as well as various reaction parameters (slope, y-intercept,correlation coefficient).
Efficiency
A PCR efficiency of 100% corresponds to a slope of 3.32. Ideally, the efficiency (E) of a PCR reaction should be 100% but experimental factors such as the length, secondary structure, and GC content of the amplicon can influence efficiency
The probe-based technique is sensitive enough to detect SNP and can distinguish between homozygous wild type, heterozygous and homozygous mutant alleles by virtue of the dissociation patterns produced.
Absolute Quantification
Requires the construction of an absolute standard curve for each target
The standard curve is based on a serial dilution of a sample with known copy number
Ct of each standard sample is plotted against the logarithm of the known concentration The standard curve is then used to estimate concentrations of unknown samples
Relative Quantification
Housekeeping gene: Abundantly and constantly expressed gene. Expression level of these genes remains constant. eg 18 S rRNA, GAPDH, Actin Normalization: To accurately quantify gene expression, the measured amount of RNA from the gene of interest is divided by the amount of RNA from a housekeeping gene measured in the same sample to normalize for possible variation in the amount and quality of RNA between different samples.
Comparative Ct Method.
This involves comparing Ct values of the samples with a control or calibrator such as a non-treated sample. The Ct values of both the calibrator and the samples are normalized to an endogenous housekeeping gene.
REALTIME-PCR APPLICATIONS
Detection of Pathogens
A Scorpion Probe Based Real-Time PCR Assay for Detection of E. coli O157:H7 in Dairy Products.
RTi-PCR method based on Scorpion probe targeting the eae gene of E. coli O157:H7.
SNP Genotyping
Multiplex TaqMan assay for SNP genotyping
Methylated DNA having C will have a higher melting temperature than unmethylated DNA having T at same position. This can be detected by melt curve analysis.