ACADEMICS & RESEARCHERS

From a Researcher, for a Researcher...

To pursue research without the risk of mutagenicity, toxicity, carcinogenicity!

Molecular biology research has been flooded with mutagenic, carcinogenic and toxic nucliec acid dyes as visualization and detection agents. Students and researchers are constantly exposed to such biohazards. A safe and food grade nucleic acid dye such as tinto rang™ would prove to be a boon to the student and research community!

Developed from a plant source, tinto rang, is the new generation fluorescent nucleic acid stain designed in India as an alternative to the highly toxic Ethidium Bromide (EtBr). Extensive scientific studies show that tinto rang™ does not exhibit mutagenicity or any other kind of toxicity. It is the safest and the only certified food grade nucleic acid dye available in the market today.

The concentration of the target molecules in the samples are reported by fluorescent dyes, which emit a signal only when bound to the targets that minimize the effects of contaminants— including degraded DNA or RNA— on the results.

Researchers uses different techniques of gel electrophoresis as per their areas of research / application:

Gel Electrophoresis:

Electrophoresis is the method of separation of macromolecules or their fragments based on size and charge. The gel can be made up of either agarose, polyacrylamide or starch. Agarose gel is used to separate nucleic acids, while polyacrylamide and starch are used for separating proteins.

Agarose gel electrophoresis:

Agarose gels are made up of natural polysaccharide polymers extracted from seaweed of genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis has revolutionized the separation of DNA and can be used to separate nucleic acids ranging from ~50bp to several mega bp, to calculate the molecular weight. Due to the uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight.

  • Gel staining:- The dye is added to the molten gel and allowed to set.

  • Pre DNA staining:- The dye is added to directly to the DNA samples.

  • Post staining:- The dye is added to the buffer and the post run gel is immersed into it.

The rate of migration of a DNA / RNA molecule through a gel is determined by the following factors:

  • Size of DNA / RNA molecule:

    Size is inversely proportional to the distance travelled.


  • Agarose concentration / Pore size

    Larger molecules are resolved better using a low concentration gel (large pore size) while smaller molecules separate better at high concentration gel (smaller pore size).


  • DNA conformation

  • Voltage applied

    The rate of migration of the DNA is proportional to the voltage applied, i.e. the higher the voltage, the faster the DNA moves. The resolution of large DNA fragments however is lower at high voltage.


  • Concentration of nucleic acid stain (Eg: EtBr dye):

    Dye like Ethidium bromide which intercalates into circular DNA can change the charge, length, as well as the superhelicity of the DNA molecule, therefore its presence in gel during electrophoresis can affect its movement.


  • Type of agarose (Eg: Low, medium and High EEO)

  • Electrophoretic time:

    The gel must be run until the band of interest has migrated to 40-60%, the length of the gel. Resolution may be decreased in smaller gels.


  • Electrophoresis buffer:

    Various parameters of the buffer can affect the electrophoretic mobility of the sample in various ways.
    Composition
    Ionic Strength
    pH

  • Capillary Gel Electrophoresis (CE):

    The currently used dyes in Capillary gel electrophoresis, have higher detection limits, which would require high amounts of DNA with higher run times and lower resolutions. Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom's radius.The rate at which the particle moves is directly proportional to the applied electric field--the greater the field strength, the faster the mobility. Capillary electrophoresis is used most predominately because it gives faster results and provides high resolution separation.


  • Polyacrylamide Gel Electrophoresis (PAGE):

    Polyacrylamide gels can be used for the separation and analysis of proteins and relatively small nucleic acid molecules. The resolution of the PAGE method is so high that, in the size range of about 10-1000 nucleotide units, it is capable of separating DNA molecules that differ in length only by a single monomer unit. In the case of single-stranded DNA, individual molecules are separated solely based on their length. This is due to the fact that, in the case of DNA (or RNA), the number of negative charges is a simple linear function of the number of monomer units (i.e. the length of the molecule). In other words, the specific charge (number of charges per particle mass) is invariant, i.e. it is the same for all DNA molecules. It is so because each monomer unit has one phosphate moiety that carries the negative charge. When an appropriate denaturing agent, such as urea, is added to the DNA sample and the gel is heated, the shape of the varying-length linear DNA molecules becomes identical. As a consequence, denatured molecules will be separated exclusively based on their size.


  • Pulsed-field Gel Electrophoresis (PFGE):

    This technique is relatively similar to a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions. With periodic changing of field direction, the various lengths of DNA react to the change at differing rates and each band will begin to separate more and more, even at very large lengths. PFGE may be used for genotyping or genetic fingerprinting but, commonly considered a gold standard in epidemiological studies of pathogenic organisms.

tinto rang is a Minor Groove Binder / External Binder (MGB/EB), it facilitates the reusability of the genetic material without altering the structure of DNA for further downstream studies /applications. tinto rang™ improves cloning efficiency. Post staining with tinto rang™ is only for about 1-5 minutes, that saves incubation time compared to other stains. The Stain can detect as little as 0.5 ng of DNA / RNA sample.



tinto rang™ in Agarose Gel Electrophoresis
E. coli plasmid DNA concentration ranging from 100 ng - 600 ng stained with tinto rang™ by in gel, with sample and post staining, and analyzed using GE ImageQuant LAS-4000 GelDoc System.
NA-Staining
Alkaline / Urea agarose gels are run at a pH that is sufficiently high to denature double-stranded DNA. The denatured DNA is maintained in a single-stranded state and migrates through the alkaline gel as a function of its size. Detection of ssDNA concentrations ranging from 500-250ng using tinto rang™ has been stained and analyzed using GE ImageQuant LAS-4000 GelDoc System.
ssDNA
Staining of tinto rang™ with plasmid DNA. Samples of DNA were resolved on 0.8 % agarose gel in TAE buffer that was post stained with tinto rang™. The gel was run at 100 volts for 1 hour.
Fig (1): E. coli plasmid DNA concentration ranging from 10-200ng stained with tinto rang™, and analyzed in GE ImageQuant LAS-4000 GelDoc System with and without Exposure settings.
Fig (2): pET Duet vector stained with tinto rang™, and analyzed in GE ImageQuant LAS-4000 GelDoc System.
plasmid-DNA
petplasmid
We have examined the effect of dye (nucleic acid stain) on E. coli Genomic DNA which was stained with tinto rang™ and analyzed using GE ImageQuant LAS-4000 GelDoc System.


Plant-genomic-DNA
Plant Genomic DNA concentration ranging from
50-150 ng stained with tinto rang™, and analyzed
in FujiFilm LAS-4000 GelDoc System.
<i>E. coli</i>-genomic-dna

Genomic DNA from E. coli with concentration of 3251 ng/ul
stained with tinto rang™, and analyzed in FujiFilm LAS-4000
GelDoc System.
Gel image showing E. coli RNA concentration ranging from 40 ng to 120 ng & 50 to 200 ng seprated as 23S, 16S and 5S upon post staining with tinto rang™ (3-5 ul of 1x in 50 ml TBE), analyzed by using GE ImageQuant LAS-4000 GelDoc System.
rna-tinto
Gel image showing E. coli RNA concentration ranging from 40 ng to 320 ng seprated as 23S, 16S and 5S upon post staining with tinto rang™ (right) and EtBr (left) when loaded with sample (1 ul dye/5 ul sample) and analyzed by using GE ImageQuant LAS-4000 GelDoc System.

Target Amplification

The Polymerase Chain Reaction (PCR) is a method of replicating DNA, it makes numerous copies of a specific segment of DNA quickly and accurately. It is capable of taking a small amount of DNA or even single molecule and amplifying a specific region exponentially such that once the reaction is finished, there may exist up to 230 copies of each starting DNA molecule. Before the development of PCR, the methods used to amplify, or generate copies of recombinant DNA fragments were time-consuming and labour-intensive. But PCR reactions can complete many rounds of replication and produce billions of copies of a DNA fragment only in few hours. We have examined the gel result stanied with tinto rang™ of PCR product (560bp) amplified using Applied Biosystems 2720 Thermal Cycler using GE ImageQuant LAS-4000 GelDoc System.

The Basics of PCR Cycling:

The three major steps in a PCR cycling reactions, which are repeated upto 20 to 40 cycles. It is always done on an automated thermo cycler(PCR), which has ability to heat and cool the reaction tubes in a very short period of time.

Denaturation (95°C), 30 seconds:

During this stage, the double strand melts open to form single stranded DNA, all enzymatic reactions stop.

Annealing (55–60°C), 30 seconds:

Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds formed are so strong that the primer stays attached.

Extension (72°C), time depends on product size:

The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3'side, reading the template from 3' to 5' side, bases are added complementary to the template).

Applications of PCR

Classification of organisms Genotyping Mutation detection Sequencing Detection of pathogens DNA fingerprinting Drug discovery Genetic engineering Molecular archaeology and many others.
PCR
pcr-sig-K2
Reverse transcription polymerase chain reaction (RT-PCR), a variant of polymerase chain reaction (PCR), is a technique commonly used in molecular biology to detect RNA expression with enzyme reverse transcriptase. Thermostable DNA polymerases used for basic PCR require a DNA template, and as such, the technique is limited to the analysis of DNA samples. Yet numerous instances exist in which amplification of RNA would be preferred. To apply PCR to the study of RNA, the RNA sample must first be reverse transcribed to cDNA to provide the necessary DNA template for the thermostable polymerase. This process is called reverse transcription (RT), hence the name RT-PCR. Visualisation of the RT-PCR product stained with tinto rang™ after amplification using Applied Biosystems 2720 Thermal Cycler has been observed under GE ImageQuant GelDoc System.
RT PCR