real-time PCR
 
 
If I use ABI Intruments, what are the reagent I could be choose?

Invitrogen provide the reagent for entries of ABI and any instrument. ABI real-time PCR you can use the reagent that specify for ABI only


 

Which housekeeping genes for qRT-PCR control?

Depend on your laboratory general to be used such as GAPDH, -actin, Elongation Factor, 16S, -globin etc.


 

How to calculate the copy number of gene?

How many copies are in a certain amount of human genomic DNA?

1 genome copy = 3 x 109 bp

1 bp = 618 g/mol

1 genome copy = (3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol

= (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 (Avogadro’s number))

=3.08 x 1012

Each somatic cell has 6.16 pg of DNA (sperm and egg cells have 3.08 pg). There is one copy of every non-repeated sequence per 3.08 pg of human DNA. Therefore, 100 ng of genomic DNA would have:

(100,000 pg of DNA )/3.08 pg =~33 000 copies 1 ng of DNA has 330 copies


 

Can I use the reagent from invitrogen with other real-time PCR

Yes, because invitrogen provide the reagent for all qPCR and qRT-PCR both SYBR Green system and probe system (Taqman, scorpion and molecular beacon)


 

What are quenchers and why are they used in Real-Time PCR? Are quenchers required when using LUX primers? Replica Handbags uk

Quenchers are moieties attached to primers or probes that can quench the emission from a fluorophore that is also attached to that probe. They usually do this by FRET (Fluorescence Resonance Energy Transfer). Quenchers are generally used in probe-based assays to extinguish or change the wavelength of the fluorescence emitted by the fluorophore when both are attached to the same oligo. When the fluorophore gets excited it passes on the energy to the quencher, which emits the light at a different wavelength (higher wavelength). TAMRA is an example of a quencher. Other quenchers, known as dark quenchers, capture the fluorescence of the fluorophore but give off heat instead of a fluorescence emission. This is advantageous because there is less contaminating fluorescence that could possibly be detected in a qPCR assay and contribute to background. Dabcyl, Eclipse, and Black Hole (BHQ) quenchers are dark quenchers. LUX primers do not need a quencher because they form a hairpin loop that quenches the unextended primer.


 

How do LUX primers work and what are the major advantages of LUX primers over TaqMan probes?

 LUX primers are currently being produced with FAM, JOE, TET, HEX, and Alexa Fluor 546 dyes, so as long as your machine detectes these dyes, you can use the LUX primers. Specific protocols have been developed for using LUX Primers on the following real-time quantitative PCR instruments: ABI PRISM® 7700, ABI PRISM® 7000, Bio-Rad iCycler™, Cepheid Smart Cycler®, Corbett Rotor-Gene 3000™, DNA Engine Opticon® 2, Roche LightCycler®, and the Stratagene Mx3000P. For protocols, please view our LUX™ Primers Instrument Protocols found online at:

http://www.invitrogen.com/content/world/instrument_protocols.pdf


 

How do LUX primers work and what are the major advantages of LUX primers over TaqMan probes?

You can routinely detect 10 copies or even fewer with well optimized reaction conditions. You can also detect picogram amounts of DNA or RNA from human genes. The dynamic range is about seven orders of magnitude or detection from 10 copies to 10,000,000 copies.
 
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Do LUX primers have more specificity than Taqman or other methods of detection for Real Time RT-PCR or qPCR?

LUX primers have greater specificity than SYBR green. However, compared to Taqman, LUX has the same level of specificity or in some cases slighly lower specificity than Taqman. Both of these systems have sequence specific probes, so they are specific to the gene that is being examined. However, Taqman assays require an additional target specific probe in addition to the primers. Therefore, in a few cases, a TaqMan assay may be more specific than LUX. The advantages of LUX primers over Taqman probes are that LUX is more affordable because an additional probe is not necessary and a melting curve analysis can be performed to verify specificity. Additionally, only one primer is labeled with a dye when using the LUX system.


 

Can you do melting curve analysis in Real-Time PCR after using TaqMan probes?

The fluorophore in a TaqMan probe is not incorporated in the new PCR product. Therefore, in a melting curve analysis, there is no change in fluorescence whether the PCR product is single-stranded (after melting) or double-stranded (before melting). On the other hand, SYBR Green I allows melting curve analysis because it fluoresces when bound to the minor groove of double-stranded DNA. When PCR products melt, they release the SYBR Green I into solution and it no longer fluoresces. PCR products made using LUX primers from Invitrogen can also be subjected to melting curve analysis because the fluorophore only fluoresces when it is incorporated into a double stranded DNA amplicon.


 

How do LUX primers work and what are the major advantages of LUX primers over TaqMan probes?

LUX™ Primers are oligonucleotides labeled with a single fluorophore, custom-synthesized according to the DNA/RNA of interest. Typically 20-30 bases in length, they are designed with a fluorophore close to the 3’ end in a hairpin structure. This configuration intrinsically renders fluorescence quenching capability; no separate quenching moiety is needed. When the primer becomes incorporated into double-stranded PCR product, the fluorophore is dequenched, resulting in a significant increase in fluorescent signal. This signal increase is the basis for the LUX™ detection platform. They differ from TaqMan probes in 1) Cost: With LUX you need two primers, only one of which is labeled. With TaqMan you need two primers. TaqMan also requires a third probe that is labeled with both a dye and a quencher. Therefore LUX primers are less expensive. 2) Melting Curve analysis: Because the LUX primer fluorophore gets incorporated into the PCR product, you can do a melting curve analysis after the PCR while the TaqMan fluorophore is not incorporated into the PCR product so a melting curve cannot be performed.


 

What is the sybergreen concentration in real-time PCR.

When you add SYBR Green I to your PCR reactions, you will need to add more magnesium. Typically, an increase from 1.5 mM to 4 mM magnesium is required for 1x SYBR Green I, but it may be ueful to titrate in the amount your reactions need.


 

What is the optimal size of PCR products and primer design considerations for quantitative PCR (qPCR)/real-time PCR?

Primers must generate a single band with no primer dimer formation (other than in the no-template control). For RT-PCR they should not amplify genomic DNA or processed pseudogenes. The general consensus for product size is from 80 - 200 bp. Shorter PCR products are more efficiently amplified, however PCR products as long as 500 bp can still be used. When comparing the relative expression of multiple genes in a single experiment, it is crucial that each gene is amplified with the same efficiency, therefore the product sizes should not be drastically different. Sometimes it is also necessary to vary the ratio of forward and reverse primers to equalize the efficiencies of different sequences. With SYBR Green I detection, larger products will fluoresce more intensely than smaller ones.


 

How do you control for carry-over contamination during quantitative PCR?

PCR products from previous PCR reactions need to be destroyed so that they do not act as templates in new reactions. In order for selective degradation of previous carryover product to occur, a GUAC dNTP mix is employed rather than the standard GTAC mix. Resulting product then contains uracil. Our Platinum qPCR Supermix UDG systems utilize this GUAC mix along with uracil-N-glycosylase to degrade only those DNA products that contain uracil bases carried over from a previous reaction. The degradation reaction occurs at 45oC before the PCR reaction. At this point any non-specific product that is created while you set up the PCR mixes will also get degraded. The glycosylase is inactivated completely at the 94oC denaturation step at the beginning of PCR and will not affect your desired PCR products.


 

The difference between conventional PCR and real-time PCR

Real-Time chemistries allow for the detection of PCR amplification during the early phases of the reaction. Measuring the kinetics of the reaction in the early phases of PCR provides a distinct advantage over traditional PCR detection. Traditional methods use Agarose gels for detection of PCR amplification at the final phase or end-point of the PCR reaction.


 

Real-Time PCR Applications

• Viral Quantitation
• Quantitation of Gene Expression
• Array Verification
• Drug Therapy Efficacy
• DNA Damage measurement
• Quality Control and Assay Validation
• Pathogen detection
• Genotyping


 

How to design primers for real-time PCR?

* Generally, primers used are 18-28 nt in length. This provides for practical annealing temperatures.

* Primers should avoid stretches of polybase sequences (e.g. poly dG) or repeating motifs – these can hybridize inappropriately on the template.

* Aim for 50% GC content. High GC content results in the formation of stable imperfect hybrids while high AT content depresses the Tm of perfectly matched hybrids.

* If possible the 3ด end of the primer should be rich in GC bases (GC clamp) to enhance annealing of the end which will be extended.

* Inverted repeat sequences should be avoided to prevent formation of secondary structure in the primer which may prevent hybridization to template.

* Sequences complementary to other primers used in the PCR should be avoided so as to prevent hybridization between primers (primer dimers).

* Primer pairs should have compatible Tm (within 5 degrees). * When adding sequences to the 5ด end of the primer to create a restriction site, it is important to include a few extra bases (2-6 bases) to serve as a clamp to keep the 5ด ends from breathing during digestion.


 

What is real-time PCR?

In Molecular Biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain reaction, is a laboratory technique used to simultaneously quantify and amplify a specific part of a given DNA molecule. It is used to determine whether or not a specific sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (Q-PCR), itself a modification of polymerase chain reaction. Each technique uses some kind of fluorescent marker which binds to the DNA. Hence as the number of gene copies increases during the reaction so the fluorescence increases. This is advantageous because the efficiency and rate of the reaction can be seen. There is also no need to run the PCR product out on a gel after the reaction.


 

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