Term
| Classic Hereditary Hemochromatosis (Type 1)- OMIM |
|
Definition
|
|
Term
| Three most common HFE mutations |
|
Definition
C282Y (c.845G>A or p.Cys282Tyr)
H63D (c.187C>G or p.His63Asp)
S65D (c.193A>T or p.Ser65Asp) |
|
|
Term
| Capillary Electrophoresis results for C282Y and H63D |
|
Definition
Wild Type: 146 bp
C282Y +/-: 146, 117 bp
C282Y -/-: 117
Wild Type: 141 bp
H63D +/-: 208, 141 bp
H63D -/-: 208 bp |
|
|
Term
|
Definition
Quantitative PCR- PCR reaction where product accumulation can be monitored as the reaction proceeds.
3 phases of PCR Reaction:
1) Exponential 2) Linear 3) Plateau |
|
|
Term
Fluorescence Detection Methods
|
|
Definition
a) Intercalating Dyes: non-specific, cheaper than probes, does not require probe design. Cons= reduced analytical specificity, bind all ds DNA, binds primer dimers.
b) Incorporation of allele specific fluorescent probes: third oligonucleotide in PCR reaction. higher analytical specificity, can be used for multiplex assays. Cons= expensive, challenges in new assay development. |
|
|
Term
Probes used in Real Time PCR Detection methods
|
|
Definition
1. Hybridizing probes: probe needs to hybridize to template to produce fluorescence eg.Hairpin probes.
2. HybProbe (Pair of probes): Self-fluorescing amplicons- fluorescently labeled PCR primer. Quencher and fluorophore, as primer replicated by polymerase, quencher is separated from fluorophore and fluorescence is generated.
3. Hydrolyzing probes: probe needs to be digested to produce fluorescence by separating quencher from fluorophore. |
|
|
Term
|
Definition
| Fluorescence Resonant Energy Transfer |
|
|
Term
| Quantification of RT PCR Results |
|
Definition
Can prepare standard curve results from dilution series of controls. By comparing Ct of test sample to standard curve, the inital DNA amount can be estimated.
Initial amount DNA known= absolute quantification
Initial amount of DNA unknown= relative quantification. |
|
|
Term
| Advantages of Digital PCR over Q-PCR |
|
Definition
| More accurate absolute quantification, greater analytical sensitivity and specificity, no external reference needed, superior detection of rare variants, can be used to create reference for other real time experiments requiring absolute quantification. |
|
|
Term
|
Definition
| A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis. eg. ROX dye provides an internal reference to which the reporter dye signal can be normalized. |
|
|
Term
|
Definition
| The dye attached to the 5' end of a TaqMan probe, the dye providea fluorescence signal that indicates specific amplification. |
|
|
Term
Advantages of Real Time PCR |
|
Definition
| No electrophoresis required, follows amplification as it occurs in the exponential phase rather than at the plateau phase, higher analytical specificity, homogenous reaction= nucleic acid amplification and detection occur in the same step, reduced opportunity for cross-contamination |
|
|
Term
| Disadvantages of Real Time PCR |
|
Definition
| Requires specialized thermocycler, no confirmation of PCR product identity. |
|
|
Term
| Real Time molecular clinical assay |
|
Definition
| multiplexed endpoint SNP detection based on Real Time principles; does not calculate amount of original DNA; distinguishes between three possibilities (homozygous wt, homozygous mutant, heterozygote). |
|
|
Term
|
Definition
| uracil-N-glycosylase: protects against cross-contamination from previous real time products by degrading nucleic acids containing uracil. Important since PCR products will not be un on a gel to confirm identity. |
|
|
