SYBR Green are dyes used for the detection of polymerase chain reactions (PCR). SYBR Green's main function is to bind the double stranded DNA and detect PCR product that accumulates during the PCR cycles. There are two different types of small molecules which perform the work of binding the double stranded DNA. These molecules which bond to the double stranded DNA are usually divided into two types:
Intercalators : Intercalators are planar molecules which bind to the DNA and insert between the base pairs of duplex DNA. What this does is separation of the base pairs along the helical axis. This enables them to be used as probes in studying of nucleic acids and DNA replciation. Ethidium bromide is one the most commonly used stain for duplex DNA axis used in many intercalation studies. Acridine is another intercalator,which is also known to be a human carcinogen. It causes mutations by creating an additional base on the opposite strand of the DNA.
Minor groove binders or MCBs as they are known form stable complexes with single strand DNA which allows for shorter probes in assays.
What are the essential requirements of SYBR Green for accurate PCR detection?
Whatever the binding technique may be the two following requirements have to be followed during every polymerase chain reactions (PCR) detection process:
No inhibition from polymerase chain reactions (PCR)
There should be increase in the amount of fluorescence when the binding with the DNA is going on.
How does the SYBR Green works?
Let us understand how SYBR Green works stepwise:
Firstly SYBR Green is added to the sample.
The moment it is dropped in the sample, it starts the binding procedure with the double stranded DNA present in the sample
During this PCR detection process, the target sequence is amplified by the AmpliTaq Gold® DNA polymerase due to which the amplicons or the PCR products are produced.
The SYBR Green now starts finding different double stranded DNA to proceed with the binding process and create PCR product in a similar way.
As the process progresses more and more amplicons or PCR products are created. As the SYBR Green molecule are very sensitive they bind with almost all the DNA strands in the sample and this further enhances the fluorescence intensity proportionate.
What is SYBR Green?
There are 2 types of SYBR green, The SYBR green I & SYBR green II. SYBR green I is 25 fold more sensitive then EtBr (Ethidium bromide) to nucleic acides and SYBR green II has good sensitivity to RNA ,and in staining dsDNA and ssDNA. Some of the general characteristics of SYBR Green are:
It is a membrane permeant nucleic acid dye or gel which evenly stains the DNA strands.
It gets maximally excited by 497 nm laser and in the second turn around after it has reached the peak, can be excited at 254 nm.
The DNA stained by SYBR Green-I emits fluorescent green emission.
It is comparatively more detection sensitive than Ethidium bromide and its fluorescence quantum yield too is five times (~0.8) more than the Ethidium bromide (~0.15).
SYBR Green I give best result during the post staining process although it can also be used during precasting as gel.
With SYBR Green you can get a detection limit of -60pg/band having 300 nm trans-illumination. As compared to Ethidium bromide this sensitivity is 25 times more.
It is also used during the denaturing of agarose/formaldehyde and polyacrylamide/urea gels for detecting the DNA as well as RNA strands before the prewash steps.
SYBR Green is also considered to be one of the most sensitive stains for oligonucleotides and makes way for the detection showing 50 to 100 times more sensitivity as compared to the ethidium bromide.
Why is SYBR green an alternative to Ethidium bromide?
SYBR green is used as an alternative to Ethidium bromide in the visualization of nucleic acids. SYBR Green binds directly to the nucleic acid and upon excitation with a light source, emitts a green light that can be used in the quantification and imaging of the DNA. SYBR green is less mutagenic than ethidium bromide. Though it is less mutagenic it is a chemical which can be dangerous and extreme caution should be taken while handling it as well. SYBR Green is quite expensive than Ethidium bromide, but it being very sensitive is preferred over EtBr.
What are the advantages and disadvantages of using SYBR Green?SYBR Green offers two main advantages:
Since it does not require any probe the running cost as well as the assay set up cost is really very low.
It is very useful in monitoring any double stranded DNA amplification.
One of the main disadvantages of SYBR Green is that since this stain binds with all types of double stranded DNA it also binds with the non specific sequences of the double stranded DNA stranded as well.