Polymerase Chain Reaction (PCR Overview)

Polymerase Chain Reaction is a technique that replicates very small amounts of DNA in large quantities. It is a biochemical process capable of amplifying a single DNA molecule into millions of copies in a short time. It is a very common process used in medical and biological labs.

The reaction/process is carried out in a thermal cycler, a tool that heats and cools tubes with biomaterial samples. Cooling and Heating are required for replication. The precision of the temperature system influences the exactness of the outcome.

Do you want to know more about this technology here’s the more detail about it.

The development of the Polymerase Chain Reaction method made the greatest contribution to the Taq Polymerase enzyme because it is the only enzyme that can withstand even at high temperatures. This enzyme was first isolated from the Thermus aquaticus bacterium.

Ingredients For PCR

The materials for Polymerase Chain Reaction include:

1. DNA template fragments (Template) to be copied.
2. The role of primers in pcr is defining both ends of the replication start point.
3. DNA polymerase (Taq polymerase).
4. Synthetic raw materials (Ribonucleotide ATCG) and buffer solution.

PCR Steps

PCR steps have major pieces involved are DNA templates, nucleotides, primers, and Taq polymerase enzyme, all these can also be considered as a building material for DNA. All such entities are synthetic  and are gathered along with cofactors required by enzyme by putting them through iterative cooling and heating steps permit the DNA to be manufactured and all this allowed to happened in the tube.

1. Denaturation: (90 ℃ -96 ℃): This step is to split the double-stranded DNA into two single-stranded DNA using the principle of high temperature breaking hydrogen bonds.
2. Annealing : (60 ℃ -65 ℃): Followed by the addition of two we can identify the specific DNA in small fragments of DNA molecule (referred to as our primer, Primer ), and at a suitable temperature of the original DNA form a bond.
3. Extension: (70 ℃ -75 ℃): At a particular temperature suitable amount of the DNA, Primer, Polymerase, dNTPs, of ATP Once interaction, respectively, by the extension of this end two primers the DNA

In a typical PCR reaction, such iterations happen 25 to 35 times which usually grabs 2 to 4 hours, all this depend on the desired extent we want to be copied of the DNA section. 

It the reaction is carried out strategically in a manner or with high expertise, then we can replicate billions of DNA sections with just a singular/sole micro piece of the original nucleac acid known as template. 

The new DNA step replicated is utilized in the ahead round-up as a template. There are certain primer replicas and taq polymerase enzyme molecules around in the reaction, so the DNA molecules quantity twice increment in each round-up. This is in-short is an exponential growth process.

What are PCR tubes

PCR tubes are little tubes manufactured for the PCR process. These tubes are available in many materials and sizes. This tube is of unique design with a tight fit cap that ensures the sample safety from contamination. Also the tube keeps the sample hydrates and saves from evaporation.

PCR Method Types:

Types of PCR are as follows:

Nested PCR:

first, amplify a few cycles with low-specific primers to increase the number of templates, and then amplify with high-specific primers.

Multiplex PCR:

The type in which more than one sequence is simultaneously amplified.

In situ PCR:

It consists of a Polymerase Chain Reaction in histological sections or cells, where the generated products can be visualized at the amplification site.

Reverse transcriptase-PCR (RT-PCR):

It is a variant of PCR in which we use RNA as the initial template instead of Deoxy Ribonucleic Acid, and it uses a reverse transcriptase (like Tth) to carry out the synthesis of complementary nucleic acid to Rinonucleac Acid.

Hot start-Polymerase Chain Reaction: 

The type of reaction with high heat-activated nucleic acid polymerase to reduce non-specific products.

Real-time PCR:

The reaction type which use a fluorescent dye or quantitative detection probes, also known as quantitative PCR, in which a plurality of sets may be compared.

Decrease Polymerase Chain Reaction:

 The temperature gradually decreases in the first few cycles.

In Silico/ Digital PCR:

Generally, PCR is used in medical and biological research laboratories in various subjects such as identification of genetic fingerprints diagnosis of hereditary diseases, diagnosis of infectious diseases, paternity test, cloning of genes, and DNA computation.

What is PCR Used For

PCR Applications include:

• Copy specific genes
• Gene performance comparison
• Gene map creation
• Detect genetic diseases
• DNA genetic evolution
• Paternity Testing
• Gene mutation research

Polymerase Chain Reaction technology is still developing. This is continuously proceeding with improvements and refinements of the procedures and devices utilized, permitting the procedure to be adjusted to address master issues. For example, new techniques and refinements are being created and utilized, particularly when the evaluation of Deoxy Ribonucleic Acid in an example is required.

What is Taq polymerase?

it is a required enzyme in Polymerase Chain Reaction. Taq polymerase is also known as DNA polymerase is used to create new strands utilizing the existing strands of DNA considering them as templates.

 Taq polymerase is isolated from the bacteria known as thermus aquaticus. This bacteria is found in hydrothermal vents & hot springs. Its polymerase is known for the heat stability and activenes of it which is about 70 degrees. 

The heat stability it possesses is an optimal range for PCR as high temperature is utilized iteratively to denature the DNA template.

What is the benefit of using taq polymerase in pcr? Taq polymerase is a notably efficient, functional, & heat-bearing enzyme when it touches its optimal temperature range. It is capable of amplifying more at opt temperature and put on 150 nucleotides/sec.

read: reagents needed for typical Polymerase chain rezction

What are PCR Primers?

Taq polymerase is useless without PCR primers as these are must-have things to make Deoxy Ribonucleic Acid by putting short nucleotide sequences that provide a starting edge for DNA manufacturing utilizing primers. 

In polymerase chain reaction the performer of the reaction figures the DNA section that will be utilized for amplification by the primer selection.

PCR primers are short entities or short chunks of single stranded DNA , generally 20 20 20 nucleotidesin extent. Primers are usually designed to haunch the greatest DNA section and most importantly 2 primers per PCR process are utilized. They are given with the sequences that will make them conjugate to strands that are opposed to Deoxy Ribonucleic Acid template, just at the copied sections to be. Primers conjugate to complementary base pairing templates.

On the time primers are conjugated to templates, they can be incremented in legth by polymerase, and the section that lies among them and will be replicated.

PCR Gel and Electrophoresis

after dealing we have to visualize results. Gel electrophoresis PCR is the process used to visualize the reaction conclusions.

It is another technique that can be utilized to that involved DNA fragments to be pulled gel matrix on the basis of their purity levels using the electric current by taking the advantage of negative charge on DNA.  A DNA ladder is generally involved so the fragment size from the PCR sequencing can be figured. 

Deoxy Ribonucleic Acid chunks of similar length create pcr bands on the gel surface which can be visualized using naked eyes in only the case gel is stained using binding dye.

Purpose of PCR / Why Do we do PCR

To have the reaction performed nicely, the native Deoxy Ribonucleic Acid strand that is going to be replicated/copied required not necessarily to be refined or in excess. 

It is most probably be a super minute particle in a material micture. So PCR is recognized to have the innumerable & widespread uses to figure out genetic disorders, bacterial & viral disorders, evolution study, DNA cloning,  and fingerprinting.

PCR is a core tool for biologists and researchers in certain types of laboratories including forensic, pathology genetics, and virology.

It can also be known with the name as PCR genome sequencing technique becaue it can be utilized to discover the unknown genome section.

PCR Technique Discover

The PCR technique as found by Kary Mullis back in 1983. Mullis was a worker in one of the very first coming biotechnology companies working for Cetus in California. 

He had experience doing work for some other scientists with the goal of producing short DNA chains. According to Mullis, he was riding hs bike and thinking about how to study mutations happens in the DNA structure when  hea understood that rather he’d created a Deoxy Ribonucleic Acid section amplification procedure. He said before his bike trip go over, he was savoring Nobel Prize prospects. Later on, he had shared the Nobel Prize with Michael Smith back in 1993.

According to Mullis, starting with the a barely single DNA molecule, the PCR on applied on this can create 100b alike molecules after just some hours of waiting. The procedure to implement is so simple and straightforward. It just needs a test tube, some reagents, and only a source of heat.

There are certain PCR advantages:

• It is a quite straight forward to perceive and take in consideration with some quick results.
• its a highly responsive technique to use with the great potential to manufacture billions of specific sequences replicas in many other advanced techniques including cloning, QRT-PCR.
• It can also be advantageous in gene level analysis in microbes, tumors and many other disorders. 
• its a powerful technique for sequence identification of various viruses that are previously undiscovered and can be helpful in further discovery of the drug of diseases caused by such viruses.
• It is the backbone of most of the researches in laboratories and a most useful and prominent practicing technique.

DNA polymerases like other enzymes are too susceptible to error,  which is a reason for mutations in fragments that are produced using Polymerase Chain Reaction.

An additional PCR limitation is even smaller Deoxy Ribonucleic Acid contamination can be raised following ambiguous or misguided results. To skip the impurity possibilities, researchers should keep back an isolated room for preparation of reagent, the PCR, and product analysis. Reagents should be dispensed into single-use aliquots. Pipettors with disposable plungers and extra-long pipette tips should be routinely used.

Samples possessing humic acids might be a factor for inhibition of PCR amplification and conduct to imprecise conclusions.

Technology Patent:

It was obviously discovered by the person who discovered this Kary Mullis and was allotted to Cetus Corporation where he was a worker. Taq polymerase pcr protein is also a patented enzyme. There have been certain high profile claims related to the procedure, counting an unsuccessful claim brought by DuPont. A Swiss pharma corporation too invested in patent rights back in 1992 and is still holding the license.  

Some relevany=t court cases to taq polymerase protein are still continued around many aspects  which are between Promega & Roche. The fact is that lawful contentions have amplified past the lives of the initial PCR and Taq polymerase licenses, which actually were expired back in 2005.

Polymerase Chain Reaction in Forensic Sciences:

Assume yourself working in a forensic laboratory. You’ve got just received a Deoxy Ribonucleic Acid test from a hair cleared out at a scene of the crime, with Deoxy Ribonucleic Acid specimen from three conceivable suspects. Your work is to look at a specific hereditary marker and see whether any of the three suspects match the hair Deoxy Ribonucleic Acid for this marker.

The marker is available in 2 forms or alleles. One possesses a sole recurrent (brown section underneath), whereas the other possesses 2 repeat duplicates. With primers the reaction response that flank the rehash locale, the primary allele manufactures  200 DNA basepair chunks, whereas the other manufactures 300 Deoxy Ribonucleic Acid basepair chunks.

In a Deoxy Ribonucleic Acid forensic test, technicians carry out conceptual analysis. So a number of distinct markers would be contrasted between the suspect’s DNA versus crime scene DNA.In a general forensic test, markers utilized are not just available in 2 distinct variations. Rather, they are extensively polymorphic, so they available in many alleles variations in very small length increments. Single STR allele may possess 20 iterations whereas the other may possess 18 and another one may just 10^^1.By analyzing numerous markers, each of which comes in numerous allele shapes, forensic technicians can construct a special hereditary “unique finger impression” from a Deoxy Ribonucleic Acid test. In general STR examination utilizing 13 markers, the chances of untrue positive are fewer than one in ten billion^¹ n spite of the fact that we may think of Deoxy Ribonucleic Acid proof being utilized to convict hoodlums, it has executed a pivotal part in absolving erroneously blamed individuals (counting a few who had been imprisoned for numerous a long time).

What is the end goal of pcr?

The very end purpose of executing this reaction is to make multiple copies of the gene of interest that is currently present in a very much small amount to study the fragment from many aspects.

How is PCR similar to DNA replication ?

Both techniques are employed to produce more copies of the DNA, these two techniques produce same dna sequence ahead from a template.

Quick Recall (what does pcr do)

• Polymerase Chain Reaction is a technology to create numbers of desired DNA replicas in a laboratory using some types of equipment and some expertise only.
• It is DNA polymerase protein thermostable dependant,  Deoxy Ribonucleic Acid primers & Taq polymerase enzyme needs design specific for the Deoxy Ribonucleic Acid interest section.
• It is iteratively executed via temperature variation phases, which permit numerous duplicates of the target DNA section to be presented.