PCR and Real Time RT-PCR under critical review

Polymerase Chain Reaction (PCR) is described as a method of “amplifying” (copying) specific sequences of DNA many times by using a PCR enzyme and artificial nucleotides.  It was originally developed for creating enough amount of DNA fragments for research purposes but later it became a tool for forensics, DNA sequencing and diagnosis of genetic diseases.

Most of us heard about PCR during Covid-19 pandemic where Real time Reverse-Transcription Polymerase Chain Reaction (Real time RT-PCR) became the golden standard for detecting Sars-Cov-2 virus, a virus which got linked to Covid-19 disease.

For Sars-Cov-2 detection, specific short sequences of the virus were selected, when the Real time RT-PCR is performed a fluorescence dye signal is released if these sequences are present in the sample, these signals get recorded by a “Real-Time PCR System”.

There are many articles criticizing PCR being an inappropriate tool for diagnosis of viral infection but, in reality, PCR itself has never been critically examined. This article will examine PCR, Real time RT-PCR, the ingredients and equipment used for their performance.

CONTENT:

• TERMINOLOGY

• POLYMERASE CHAIN REACTION
  • Short history of PCR
  • How PCR works
  • DNA Isolation
  • PCR Ingredients
  • PCR steps
  • Critical examination of PCR

• REAL TIME RT-PCR
  • How Real time RT-PCR is used for Sars-Cov-2 diagnosis
  • Real time RT-PCR ingredients and equipment
  • RNA Isolation
  • Real time RT-PCR Steps
  • Critical examination of Real time RT-PCR

• FINAL THOUGHTS AND CONCLUSION

• FURTHER INFORMATION
  • Research papers on DNA amplification
  • Research papers by Kary Mullis et al establishing the PCR method
  • Articles
  • Videos

TERMINOLOGY

I will try to limit the use of scientific terms but will be using some of the below terms in order to avoid long and complicated sentences.

• DNA amplification: artificial increase of a particular DNA fragment/sequence
• PCR: Polymerase Chain Reaction, a method widely used to amplify (copy/multiply) specific region/sequence of DNA
• RT-PCR: Reverse Transcription (RT) PCR, a method which is used when the sample consists of RNA. RT is a procedure that turns RNA into DNA by producing a complementary strand (cDNA) which will match the RNA strand
• PCR Cycle: one cycle of PCR consists of three steps, Denaturation, Annealing and Elongation. During these steps the DNA gets split and copied
• Nucleobase: base pair component, adenine (A), guanine (G), thymine (T), cytosine (C)
• Nucleotide: structural unit of DNA/RNA i.e. one nucleobase and its back bone
• Oligonucleotides: short strands of synthetic nucleotides
• dNTPs (deoxyribose nucleotide triphosphates): synthetic nucleotides
• Polymerase: an enzyme that is used for artificial synthesis of DNA
• DNA Primers: short strands of synthetic nucleotides which are used by polymerase in the initiation of DNA amplification
• Probes: Oligonucleotides dyed with fluorescent dye
• Template DNA/RNA: a single strand of DNA or RNA
• Precipitate/Pellet: solid matter in the test tube
• Supernatant: liquid matter in the test tube
• Sputum: saliva, phlegm and/or mucus that is expelled from the upper part of the respiratory tract
• BALF: Bronchoalveolar Lavage Fluid collected from the lungs
• Thermal cycler (aka thermocycler, PCR machine, DNA amplifier): a machine which is used for PCR
• Centrifugation: vigorous spinning performed by centrifuge machine

POLYMERASE CHAIN REACTION

Short history of PCR

Kary Mullis was credited with the PCR invention for which he received the 1993 Nobel Price in Chemistry. Per Mullis the idea of PCR was conceived while driving, he credited the use of recreational drugs, specifically LSD, for conceptualizing the PCR procedure. K. Mullis, H. Erlich, R. K. Saiki and their team working at Cetus Corporation have developed the PCR procedure. The PCR procedure got patented and later the patent was sold to Abbott Pharmaceuticals. There are several court disputes about PCR and Taq polymerase enzyme as a result these patents haven’t been renewed.  

Drawing of PCR by Kary Mullis

How PCR works

The current PCR procedure is quite simple, you don’t even need to be a molecular biologist or a chemist to perform it, all you need is a sample that contains DNA, many chemicals, synthetic DNA components  and a thermal cycler, with all the mentioned being sold exclusively by biotech companies like Thermo Fisher and Agilent Technologies. All you have to do is to extract DNA as per DNA extraction kit instructions, mix it with the PCR chemicals, place it in the thermal cycler, set the program (temperatures and how many cycles to repeat) and voila after an hour or two you have more DNA to examine and work with.

DNA Isolation

DNA isolation/extraction

Before performing PCR, DNA needs to be extracted from the rest of the matter. Since Friedrich Miescher’s initial DNA extraction, not much has changed in the methodology of extraction other than the introduction of special equipment, heat and tons of DNA extraction kits on the market. Each kit and equipment provides their own protocols of use.

DNA Extraction (aka DNA Isolation, DNA Purification) consists of four steps:

Cell “lysis” (i.e. break down of the cell):  the substance that contains DNA is mixed with synthetic enzymes, e.g. RNase and protease, and also with a cocktail of alkalizing and acidic chemicals which it is called “buffer”. The solution will most probably be vortexed and then incubated at warm to high temperature. Then the solution will be centrifuged (vigorously spun) in order to break the matter into “supernatant” (the liquid part) and “pellet” (solid matter which will reside at the bottom of the tube). Depending on the kit’s protocols, the DNA will be in the supernatant or in the pallet, the unneeded matter will be discarded and the kept matter might be mixed with buffers and centrifuged few additional times, the final DNA will be in the supernatant.

“Precipitation” (of the DNA from the solution):  synthetic alcohol will be added to the substance obtained from the previous step, the new solution will be incubated and centrifuged. This time the DNA will be in the pallet, the supernatant will be discarded, and DNA left in the tube will be left to be air dried.

“Wash” (the DNA): to remove any remaining cell debris, chemical alcohol will be added (again) and the solution will be centrifuged once more, the supernatant will be discarded, and the pallet obtained is considered to be the isolated DNA.

Resuspension (of the DNA): the isolated DNA will be mixed with a buffer or molecular grade water i.e. the isolated DNA will be suspending in a chemical solution, buffer, or ultra-pure water, depending on how long the DNA will need to be stored. The buffer stabilizes DNA and prevents its degradation.

PCR Ingredients

• Isolated DNA: Most of the times it will be extracted from some kind of body fluid (blood, sputum or BALF).
• Water
• PCR buffer solution: A typical buffer contains Tris-HCI (Tris(hydroxymethyl)aminomethane hydrochloride) and KCl (Potassium chloride). These chemicals are used for keeping the pH of the PCR cocktail at constant value (between 8.3-9pH) which provides a stable environment for the DNA polymerase activity
• DNA polymerase: an enzyme that synthesizes DNA strands. The most known and used one is Taq polymerase
• MgCl₂ (Magnesium chloride): boosts the activity of the polymerase and as a result enhances DNA amplification
• (NH₄)₂SO₄ (Ammonium sulfate): plays the same role as MgCl₂
• DNA Primers: Primes are short, single-stranded artificial DNA sequences that get attached to specific region of the single strand of DNA. They indicate to the polymerase the starting point of the sequence in which scientists are interested in copying. Scientists can purchase predesigned primers or they can request tailored made sequences, in both cased they can only be purchased from biotech corporations.
•  dNTPs: are artificial nucleotides which are used as building blocks by the polymerase for amplifying DNA. dNTPs are also exclusively sold by biotech corporations.

The mentioned ingriendets will be mixed and placed into the thermocycler machine. The thermocycler will apply specific temperatures for a specific duration of time several times (“cycles”) to amplify DNA.

PCR steps

PCR procedure consists of the following steps:

1. Initialization: The mixture is heated to 94-96°C from 30 seconds to several minutes. This step activates the DNA polymerase and it is done once, at the beginning of the PCR process.
2. Denaturation: The mixture is heated up at 94-98°C for 15-30 seconds. This step will “denature”, i.e. split, the DNA into two single strands.
3. Annealing: In the annealing stage the temperature will drop to 50-65°C for 20-40 seconds, this temperature allows the primers and the polymerase to bind to the region we are interested in amplifying. Usually, the perfect temperature is few degrees lower than the “melting temperature” of the primers.
4. Elongation/Extension: During this step the temperature will rise to 72-80°C for 20-40 sec. During this temperature the polymerase gets activated and starts attaching free floating dNTPs to the single strand of DNA. This step results in the creation of two new pieces of double strand of DNA. The length of this step depends on how long we want the DNA copy to be. Typically, DNA polymerase can copy 1,000 base pairs per minute.
5. Final elongation: Optional but recommended step where the temperature is held at 70-74°C for several minutes (usually the same temperature as in Elongation step). This step allows the polymerase to finish copying whatever strand it is currently working on.
6. Final hold: once the preset program is finished, the thermocycler stores the end result at a temperature that prevents degradation, 4-12°C.

Steps 2 to 4 represent one PCR cycle and this cycle gets repeated 15-40 times. The more cycles are performed the more DNA copies are generated but after specific number of cycles i.e. over 40, primers and nucleotides might run out causing problems with the final DNA in the tube and in addition many cycles reduce PCR efficiency due to accumulation of by-products.

Critical examination of PCR

• DNA: I already discussed DNA concept in my previous article which I highly suggest reading as it will help in understanding what DNA might or not be. For those who want to progress with this article and examine DNA at a later stage here is a quick summary of my main findings:
  • In the DNA isolation/extraction process, the matter of interest gets mixed with chemicals and centrifugated several times, at the end a precipitate will appear in the tube which is assumed to be DNA. My personal description of this procedure is a “chemical wash of dead tissue” rather than isolation or extraction because a proper isolation or extraction is when we remove the matter of interest from the rest of the matter without chemicals, spinning or heat treatment.
  • The extracted DNA rarely, almost never, gets observed under a microscope.
  • The components of the base pairs, adenine (A), cytosine (C), guanine (G), and thymine (T):
    • Are invisible, but these invisible components, supposedly, are what separate humans from plants, trees, frogs etc.
    • Their quantity, positioning and pairing were postulated based on “Chargaff’s Rule”. In his research paper Erwin Chargaff isolated the based pair components and concluded that the quantity of A is similar to T and quantity of C is similar G. Chargaff never suggested the pairing mechanism based on these quantities.  
  • Double helix form is suggested by applying mathematical models on DNA x-rays. Math is a form of a language, quantitative one, mathematics don’t exist in nature; in addition applying mathematical formulas on few photographs of almost invisible item, can tell very little about the actual form and structure of this item. Only two x-ray pictures of specific DNA (“DNA salt” of a calf thymus aka NaDNA) were used to determine, mathematically, the DNA structure and form, since then we have only a few photos of DNA from unknown sources and of unknown extraction methodology. The DNA form was heavily based on DNA hypothetical molecular structure and the hypothetical molecular structure was based on the hypothetical form.
  • DNA role is postulated, we don’t have any real, documented evidence showing that DNA gets copied, transcribes into RNA and RNA into protein. All suggestions are based on examining, quantitatively, a sample before and after certain procedures (chemical washes, centrifugation, heating and boiling). Microscope is rarely used.
• DNA polymerase is described as an enzyme that can withstand and operate at high temperatures. Here is a very quick summary of Mullis et al research papers where they experimented with E. coli and Taq polymerases:
  • An “isolated” (i.e. chemically treated) E. coli or Taq is mixed with ingredients obtained from biochtech corporations (dNTPs, buffers etc.). In the E. coli research paper, they even used barium salt, Dowex 50 resin and fluoride.
  • After centrifugating, heating and cooling the amount of participate obtained is measured by various fractional precipitation techniques (E. coli research paper) and by gel electrophoresis (Taq research paper) to determine the “molecular size/weight” of the “DNA” in the participate.
  • There is no actual observation of enzymatic activity and some DNA turning into more DNA. Documenting certain quantities of byproducts after mixing chemicals with dead tissues or bacteria do not prove that there is an enzyme (polymerase) that gets attached to primers and to the single strand of DNA (ssDNA) and somehow, magically, connects free floating dNTPs to the ssDNA at 72-80°C.
  • Enzymes are known for digesting and degrading matter, not for synthesizing.
  • Control experiments without the polymerase were not carried out, neither without dNTPs or other chemicals to establish the effect of each component in the chemical cocktail. 
• We really don’t know how artificial nucleotides (dNTPs)are synthesized, the exact ingredients and methodology are not publicly disclosed, and they are considered trade secrets. The only way to obtain them is to purchase them from biotech corporations and trust that we have received what we have paid for, as there is no way to validate or confirm the content received.  Something very interesting is that when buying them there is always a warning, “For Research Use Only. Not for use in diagnostic procedures“ i.e. if they are used for diagnosis no one can take a legal actions against the biotech corporations in case there is a problem with the product. This clause is applicable almost for all ingredients and tools used for PCR by all biotech corporations.
• Primers are also bought from biotech corporations. Primers development is based on Oligonucleotides “synthesis cycle”, this procedure contains several steps and chemicals like acetonitrile, 3% trichloroacetic acid in dichloromethane, argon, N-methyl imidazole, pyridine and iodine. It is quite awkward that primers/oligonucleotides need so many steps, chemicals and solvents to get synthesized and all that without a microscope’s observation and some kind of control experiments. Because primers are invisible what we will actually receive is an empty bottle and a quality assurance sheet and as with dNTPs we cannot validate or confirm that in the bottle received we have primers and if there are any primers in the bottle that they are the sequences we have ordered.  
• Why Primers get attached on their own but dNTPs needs an enzyme? Do they contain some kind of a “glue” ingredient? if that is the case why not to add that glue to dNTPs (?) this can eliminate the need of polymerase.
• During Denaturation the PCR cocktail gets heated up to 95°C. Heat is very damaging to the tissue, to cells and the content of the cells. While radiation, a form of heat, is scientifically acknowledged to induce apoptosis and DNA damage, in case of PCR it is postulated that the temperature of 95°C splits the DNA into two stands. Why 95°C heat splits DNA into two strands and not into various fragments? e.g. single or double fragments? Since molecular biology doesn’t use microscope and DNA is extremely rarely studied under microscope, what evidence do we have that DNA splits into two strands at the mentioned temperature? Someone might argue that this is why those stabilizers (chemicals) are used, but how exactly can alkaline/acidic chemicals stabilize something at 95°C? Heated chemicals can actually cause more damage to the matter under study. 
• At the Annealing step, brainless synthetic primers get attached to the single DNA strands at 60°C. This step doesn’t make any sense, just because we mix dead tissue with synthetic analogue (artificial nucleotides) that might match, doesn’t mean they get attached, especially in a mixture where chemicals are the majority of the content. For example, if you place a large and small piece of the same fabric and some acids in a pot and heat it up to 60°C these pieces won’t bind and become one again, neither chicken cut out into pieces will get assembled during soup cooking. I know the comparisons sound kind of funny, maybe even idiotic, but there is no actual evidence that primers get attached to DNA at 60°C, or primers doing anything at all. If annealing temperature is the temperature of attaching, then why don’t the split DNA strands get reattached to each other since they match perfectly? Synthetic short strands of DNA get attached but original DNA strands don’t?
• The temperature of the annealing needs to be 5-7°C less than the “melting point” of the primers. The melting point is defined as “the temperature where the 50% of double-stranded DNA (dsDNA) is split into single-stranded DNA (ssDNA)“.  There are several methods of calculating the annealing temperature with each method suggesting different temperature.  This temperature is mainly based on the nucleobases, specifically on the hypothetical molecular structure and temperature “assign” to each nucleobase. “A Phenomenological Model for Predicting Melting Temperatures of DNA Sequences” article provides information on the many assumptions made when trying to calculate primers melting point temperature. If the temperature is not right then the primers, supposedly, won’t get attached. Now here is a critical question: What is the link between the two data? (1) Primers get attached at a temperature where (2) 50% of dsDNA turns into ssDNA? Primers are already single stranded, and dsDNA already got split in the denaturation step.  
• At approximately 75°C the polymerase becomes active and “amplifies” DNA. Per PCR description the polymerase recognizes one of the ends of the primer and the next missing base pair of the DNA and it, somehow magically, picks up the floating around synthetic dNTPs and places them accordingly to “Chargaff’s base paring rule”. The only way I can visualize this step is by imagining polymerase with three hands, one holding the single strand of DNA, the other the primer and the third hand is picking up free floating synthetic nucleotides, not just any that is close by, but the right one that matches the Chargaff’s rule. Polymerase knows the direction of the DNA replication (from 3’ to 5’) that is why it gets attached to the right end of the primer, it also knows from where the replication should start, because there is this rule on how DNA gets copied, from 3’ to 5’ direction (another non established assumption). It doesn’t move backwards, i.e. from 5’ to 3’, and it doesn’t attach to the other end of the primer, it knows, it operates based on molecular biology established rules (which have never been observed under microscope or validated). The reality is that again we have no evidence of anything like that happening. The original research papers isolating and describing polymerase role and action are based on matter being chemically treated, heated, and the volumes of different products before and after being compared, conclusions were based on statistically analyzing the byproduct generated after these steps e.g. there is an increase in radioactivity or florescence in the end result so this means polymerase synthesized more DNA.
• In the scenario where the primers don’t get attached then polymerase will not copy? It only copies if primers get attached? Is it that smart? What will happen if PCR is performed without primers? Did they perform such an experiment? and if yes what is the end result?
• Tris-HCI is generated by the reaction of mixing chemicals, specifically synthetic alcohol, polyoxmethylene (formaldehyde-based plastic), nitromethane (generated by combining propane and nitric acid in the gas phase at 350–450°C) etc. This mixture supposedly maintains the pH of the PCR cocktail at constant value. I’m not aware of anything living to continue to live (e.g. DNA, polymerase enzyme) when extracted from the source of nutrients and placed in a chemical solution. Based on the ingredients Tris-HCI looks more like a preservation solution rather than stabilizer, if that is the case then how exactly polymerase (an enzyme obtained from E.coli or Thermus aquaticus bacteria) operates in such (heated) acidic solution?
• MgCl₂ (Magnesium chloride) is an inorganic salt which increases the acidity of the mixture. Nothing living, e.g. polymerase, can actually “live” or “operate” in a such a chemical environment especially when heated up.
• Ammonium sulfate is “potentially dangerous to people and the environment” but it is perfectly fine for the DNA amplification.
• Let’s examine the Thermal cycler machine and its operation. The temperature between each stage in the Thermal cycler changes rapidly, each step of the cycle lasts less than a minute, how exactly does the chemical cocktail cools down from 95°C to 60°C and then after less than a minute again rises rapidly to 72°C-80°C? The Thermal cycler operates on electricity, lets imagine it is an electric oven, it uses metal plates to conduct heat. Even if the thermal cycler manages to regulate its temperatures fast enough, the content in the sample tubes will require some additional time to adjust and drop or increase to the temperature required for each specific steps. Because we are talking about seconds, I personally doubt that the content in the test tube manages to drop for annealing stage and then rise for the elongation stage. In addition, how fast exactly an electric oven or the thermal metal plate can actually change its temperature (?) especially decreasing the temperature in seconds? Does it apply some kind of cooling mechanism? Is it some kind of an oven and freezer in one? Is it possible having these two in one (?) in such a small piece of machine? Let’s examine QIAamplifier 96 which promises “Fast cycling protocol, down to 45 minutes for 30 cycles” and let’s use the shortest cycle possible:
  • 45 minutes = 2,700 seconds
  • 30 short cycles = 1,500 seconds = (15sec + 20sec + 15sec) x 30
  • 1,200 seconds are left for the adjustment in temperatures, 1,200/30 cycles = 40sec for each cycle
  • Each cycle has 3 steps which means 13.3sec for each transition, for each temperature change
  • I seriously doubt that the necessary temperature can be reached, especially dropped within 13.3sec by the thermal cycler and the content in the sample tubes or by any equipment operating with heat.
• The original steps of PCR cycle were performed manually and the ingredients of each step were added before each step of the cycle. Now all the ingredients are added in advance and all go through all the cycle temperatures multiple times. Just a critical thought, heating this PCR cocktail won’t have a degrading effect on some, if not on all, ingredients? Like seriously, every time during denaturation, we get two perfect single strand of DNA? Is it possible to expect something new to be produced in this hot chemical soup?
• There is absolutely no microscopical examination of the isolated DNA before and after the chemical treatment, after each temperature change, and after each PCR cycle. Everything is assumed that is done correctly because if scientists run a gel electrophoresis (will be covered in another article) and intense fluorescence is present then that means they have extracted high quantity and quality of DNA/RNA, regardless if they don’t know what they have ended up with in the tube as (mentioned many, many times already) molecular biologists don’t use a microscope. Someone can tell me that scientists can sequence the end result in order to confirm and study it but sequencing machines and ingredients are quite expensive, not many laboratories can afford them plus sequencing ingredients and technique are also as questionable as DNA isolation and PCR process.
• When someone searches about PCR, he/she will get animated pictures and videos on what happens in the sample tubes, but the reality is that there are absolutely no valid and visual evidence of each step happening as animated i.e. during 95°C the DNA splits, at 60°C primers get attached and at 75°C the DNA gets “copied”. We do not know if DNA splits at 95°C, we do not know if primers get attached and we do not know if polymerase attaches dNTPs to DNA strands. There are absolutely no evidence of any step actually happening as described.
• Last but not least the amount of chemicals used, for DNA isolation and in the PCR procedure, is beyond comprehension, especially if we consider that primers and dNTPs are also chemicals since they are artificially designed using chemicals. The amount of some (dead) tissue we are interested in is less than 1% of the solution, maybe even less than 0.1%. For some reason I believe that by, simply, studying the tissue under microscope without all the chemical treatment and heating can tell much more than all the mentioned.

Real time PR-PCR

How PCR is used for Sars-Cov-2 diagnosis

A modified version of PCR, Real time Reverse-Transcription PCR (Real time RT-PCR), is used for detection of Sars-Cov-2. This virus detection method was originally developed by Victor M Corman, Christian Drosten et al. and it consist of some additional ingredients, equipment and steps.

The additional steps of Real time RT-PCR are:

• Sars-Cov-2 is declared to be a RNA virus (single strand), because PCR technique was developed based on and for DNA (doble strand), the RNA needs to be turned into DNA before performing PCR. This step, the turning RNA into DNA (by synthesizing the complementary strand of the RNA) is called Reverse-Transcription (RT).
• “Real time” is performed by a PCR system that contains Fluorometer. When scientists want to determine if there is a specific sequence in the sample they add fluorescence dyed oligonucleotides (aka probes). If those probes get attached to the the DNA/RNA template then a fluorescence signal will be emitted during the Elongation step. This signal will be captured by a camera and it will be recorded by PCR software. For Sars-Cov-2 detection specially designed Primers and Probes are used, these Primers and Probes are based on short sequences of Sars-Cov-2 genome which was shared on Genbank and Gisaid.

Real time RT-PCR ingredients and equipment

• Isolated RNA
• Buffer and chemicals (same as regular PCR): Tris-HCI,  KCl, MgCl₂ and (NH₄)₂SO₄
• Reverse transcriptase (aka RNA-directed DNA polymerase): an enzyme that turns RNA into DNA. It synthesizes the complementary strand of RNA (cDNA)
• Random Hexamer Primer: these primers are used for synthesis of cDNA
• Sars-Cov-2 Primers: oligonucleotides based on Sars-Cov-2 genome
• DNA Probes (aka TaqMan) for Sars-Cov-2: Oligonucleotides based on Sars-Cov-2 genome which contain fluorescence dye
• DNA polymerase
• dNTPs (artificial nucleotides)
• Real-Time PCR System: is system that combines Thermal cycler, Fluorometer and specially designed software. Real-Time PCR system not only copies DNA but it also records the release of fluoresced signals if fluorescence dyed probes get triggered.

RNA Isolation

As with typical PCR where DNA needs to be isolated, before performing RT-PCR the RNA also needs to be isolated. The steps of Sars-Cov-2 RNA isolation are quite similar to DNA isolation. A swab with the collected sputum is placed into a reagent solution, the RNA will be extracted as follows:

• Cell Lysis: Lysis buffer e.g. trizol reagent and chloroform are mixed with the solution containing RNA. The mixture will be shaken or vortexed and centrifuged. The upper layer (aqueous layer), of the solution, which contains RNA, will be transferred to a new tube.
•  Precipitation: Synthetic alcohol (ethanol, isopropanol or isopropyl alcohol) will be added to the RNA solution, the mixture will be centrifuged, and the supernatant (the liquid part of the solution) will be discarded. The RNA will be in the pellet, this step will be repeated one to two times.
• Resuspension/Elusion: The obtained RNA will be resuspended in a water, synthetic alcohol or in a buffer.

Real time RT-PCR Steps

• The isolated RNA will be mixed with Reverse transcriptase enzyme (RT) and artificial nucleotides. This enzyme will synthesize the complementary strand of DNA (cDNA) which will match the RNA strand.
• Denaturation step is same as with typical PCR, heating the mixture up to 95°C.
• During the Annealing step, the primers and sars-cov-2 probes will get attached to the single strands of DNA (ssDNA), the sars-cov-2 primers and probes will attach only if the RNA/cDNA contains these kind of short sequences.
• At the Extension stage where polymerase synthesizes the new strands, if the polymerase reaches an attached probe, the probe will emit a fluorescence signal (light) which will be captured by a camara and recorded by a software.
• The software will record probes signaling and their increase with each cycle.

Critical examination of the Real time RT-PCR

All critical points raised during PCR examination are relevant to Real time RT-PCR. But Real time RT-PCR contains several additional issues:

• While we have few electron microscope pictures of DNA (which are nothing more that a twisted strand of unknown source), we have zero pictures and evidence of RNA existence, its form, molecular structure and function.
• Reagent solution which is used when collecting sample containing RNA e.g. sodium azide, is a highly toxic chemical.
• Why is reverse transcriptase used and not Taq polymerase for synthesizing complementary strand of the RNA? What is the exact difference between the two enzymes?
• RT-PCR is used for RNA. In RT-PCR the RNA gets “reverse transcribed” into DNA (cDNA) using reverse transcriptase enzyme. This is based on the theory that DNA transcribes into RNA and RNA gets translated into proteins. We have no actual evidence of this theory i.e. that these processes actually happen in our cells or artificially, basically that the rarely observed under microscope, delicate DNA somehow (with the help of an enzyme) produces a single stranded DNA (RNA) and that this single strand provides “instructions” to the “ribosomes” on how to synthesize proteins.
• For some unknown reason the labs performing Sars-Cov-2 Real time RT-PCR tests don’t disclose the number of cycles performed, the only thing we get is a positive or negative result plus the primes and probes used to detect the presence/absence of the virus. Cycles number is important as the higher cycle number the higher the chance of a positive or inconclusive result. I don’t understand why such a vital information which affects the result is not disclosed especially since we pay for this test (directly or through our tax money). Isn’t it our right to know, to be informed on all aspects on how these tests are performed?
• The Sars-Cov-2 RT-PCR test developed by Victor M Corman, Christian Drosten et al has tons of issues with main ones being:
  • The primes and probes were designed based on “SARS-related virus sequences available in GenBank by 1 January 2020”, this is because no official release of Sars-Cov-2 genome was available yet. Basically, the RT-PCR got designed based on a computer generated genome.
  • The research paper was submitted on 21 January 2020 and got peer-reviewed and published within less than 24 hours (never happen before); the peer review hasn’t been released to public.
  • Drosten happens to be an editor of the journal their paper got published in (eurosurveillance).
  • TIB Molbiol, a German biotech-company, developed the Sars-Cov-2 RT-PCR kit based on Corman-Drosten protocols and started sending them out as early as 10 January 2020, that was before the official announcement of a virus by WHO and the official public share of the sequence. 
  • In addition, several genome sequences were released by chinses scientists in GISAID, GENBANK and Virological.org between 10^th and 11^th January, not much is known how these genomes were generated i.e. methodology of extraction and sequencing material and equipment used.
  • Two of the writers of the RT-PCR research papers happen to be Olfert Landt the founder of TIB Molbiol and Marco Kaiser a scientific adviser of Tib-Molbiol.
  • The amount of chemicals and procedures in RT-PCR are almost doubled when compared to PCR, which makes someone wonder what exactly ends up in the test tube other than a tiny fragment of body fluid suspending in hot chemical soup.

Final Thoughts and Conclusion

As with DNA extraction and function I’m not convinced that what is claimed is actually happening during PCR. There is no microscope involvement, control experiments are not performed, there are these questionable equipment and chemicals and tons of steps and heat.

What I personally see is an unjustified and unproven claim that a small amount of dead or degenerated matter (anything extracted from the source of nutrients is dead or in process of decomposition) when mixed with many acidifying chemicals and trade secret “ingredients” and heated up for a considerable length of time, will generate many copies of “DNA”.  

How exactly does a bacterial enzyme, isolated from thermal springs synthesize matter? Bacteria digest, ferment and decompose matter, they are natural recyclers. This bacterial enzyme (Taq or E.coli polymerase) was isolated using tons of chemicals and heat, how exactly a bacterial enzyme operates without the bacteria which produces it? And how exactly it attached synthetic nucleotides to synthetic sdDNA?

The reality looks quite simple, when common sense is applied, when we use tons of chemicals, heat and centrifugation, the only thing we will get is a toxic soup containing 0.0000001% dead matter (if we believe that PCR multiples something then it can generate 1,073,741,824 copies just from one DNA in 30 cycles) . If I needed to compare PCR or Real time RT-PCR procedures to something it would be to the heating up of a meal in a microwave or in an oven. Imagine placing a small amount of meat with plenty of chemicals and some synthetic matter that advertised to turn into synthetic meat into the oven for half an hour. Will this process create more meat (?) that can be consumed? Or we will end up with a highly toxic and uneatable mixture, might even be hazardous for the oven and the environment.

Another analogy would be mixing pieces of legos with a chemical cocktail, placing the mixture in the oven and expecting assembled legos after an hour at 95°C.  Wouldn’t they degrade or melt? Wouldn’t everything under PCR temperature simply get degraded?

In reality nothing new forms in high temperatures, either during boiling or heating etc. Matter simply get acidified and the ingredients get degraded. The claim that DNA polymerase synthesizes something might be actually a byproduct of ingredients’ acidification and degradation during heat treatment.

I feel there is no need to get into much depth and history of PCR. Someone who reads those papers and using some basic logic will understand that there is absolutely no proof that DNA polymerase attaches to anything or synthesizes anything because the chemicals used, methodology of extraction and conclusions derived are all based on volumes of liquid before and after chemical treatment, heating and centrifuging. Once again absolutely no microscope involvement.

If we remove all the biochemical products and latest biotech equipment will molecular biologists be able to perform any of their experiments? It is obvious that molecular biologists rely extensively on biotech corporations, are unable to generate their own materials/stock and are unable to validate products’ accuracy and equipments’ efficiency. These facts leads me to question the power and control biotech corporations have gained during the past decades in the field of science.

As with DNA extraction PCR is nothing more than a cocktail of chemical heated for a considerable amount of time.

We are asked not to question the discoveries and processes of science or molecular biology, they are “established”, Nobel awarded, just trust the animated images and videos we are provided with to explain what happens in the sample tube. They call it science; I personally would call it a cult. A cult that developed tons of unnecessary terms and procedures which makes their “science” unattainable by an ordinary person as a result makes people more susceptible to “trust and follow” the scientists. If a person removes the terminology barrier and looks at the ingredients and procedures as they are, he/she will realize that biochemistry or molecular biology is nothing more that measurement of byproducts generated from chemical reactions and heat, byproducts that can tell very little about life and especially about health.

Someone might ask so what turns sars-cov-2 RT-PCR “positive”? I have to admit I don’t know. What I know is that Covid-19 (the disease that is supposedly linked to the Sars-Cov-2 virus) doesn’t contain any new or unusual symptoms in order to be labeled as new/novel disease. Covid-19 contains all known symptoms of almost all common dis-eases; from symptoms of common cold and flu to pneumonia and gastroenteritis, lately blood clotting, headaches and even depression are added to the very long list of Covid-19 symptoms. Someone testing positive will be treated based on Covid-19 protocols which consist of an experimental mixture of drugs and even ventilator. Someone with symptoms who tests negative will be treated the old school way i.e. treatment and/or drugs will be prescribed based on symptoms. In addition, real time PR-PCR introduced us to a new concept, the concept of being an asymptomatic carrier of a virus that can cause death to someone else. It is an interesting concept, being a carrier of a deadly virus that causes no symptoms to the host but can kill someone else when transmitted. Imagine if this concept was introduced for the common flu, no regular person would believe it and might start questioning medical/molecular science. And how exactly is this virus transmitted? How is it proven that even if something is transmitted, it causes symptoms? Causes symptom/s to one person but no symptom/s to another? If it is found in a healthy person and in an ill person then maybe it is not the cause of the dis-ease? Were Koch’s postulates satisfied by Sars-Cov-2 (?) by any virus? Virus isolation is as unnatural and disturbing process as DNA and RNA isolations containing plenty of chemicals and steps (virus isolation and cytopathic effect will be discussed in a future post).

What surprises me is that scientists reached the stage of amplifying synthetic DNA. What exactly will they gain from copying and studying synthetic DNA or RNA? Even if those sequences are created based on some human or viral (computer generated) genome what exactly synthetic sequences can tell about humans, animals, diseases, bacteria, viruses, life in general? Imagine if scientists and the medical industry reach the stage in announcing discoveries and creating diagnostic tests, tools and equipment based on these artificial, totally unreal “sequences”.

The astonishing part is that scientists are even trying to create synthetic cells and organism based on DNA [sequences] by using PCR, although they have little to no success, they excuse the failure by limiting tech, specifically, limitations of PCR process and equipment. The scientific delusion reached such a stage that they suggest PCR to become “a method of cell replication” which they’ve labeled “Replication cycle reaction (RCR) where “…this method is expected to be a platform system for building artificial cells with the ability to replicate large DNA. Considering that RCR is a reconstituted system of genome replication from E. coli, the system would be applicable to elucidate the mechanism of genome replication and division of microorganisms. However, several technical challenges remain…” If they believe in the possibility of creating synthetic life then they must believe that we are nothing more that piece of meat being alive and operating due to chemical reactions. Are my thoughts, observations and feelings products of chemical reactions? There is no involvement of some kind of a form of consciousness? So what about of all those stories’ of out of body experience and also near the death experiences? Illusions? The sense that we are much more than a physical body is a delusion?

At this point, with all the “established” science which affects our lives on daily basis especially the past two years, “think for yourself” is our best and most important tool we possess to deal with Covid-19 governmental measures, main stream science, medicine, education and health.

Sincerely,

Tam

PS: something that surprised me is not only the amount of chemicals used but also the amount of one-use plastic e.g. sample tubes, pipet tips, gloves, synthetic alcohol etc. It looks like quite polluting science, on chemical and plastic levels.

FURTHER INFORMATION

Research papers on DNA amplification:

• 1967, Enzymatic Synthesis of DNA, XXIV. Synthesis of Infectious Phage φ X174 DNA
• 1971, Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA’s as catalyzed by DNA polymerases DOI: 10.1016/0022-2836(71)90469-4
• 1976 Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus PMC232952  

Research paper by Kary Mullis et al establishing the PCR method:

• 1982, Winkler ME, Mullis K, Barnett J, Stroynowski I, Yanofsky C. Transcription termination at the tryptophan operon attenuator is decreased in vitro by an oligomer complementary to a segment of the leader transcript https://www.pnas.org/content/79/7/2181
• 1985, Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science (New York, N.Y.). 230: 1350-4. PMID 2999980 DOI: 10.1126/Science.2999980 
• 1986, Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia On Quantitative Biology. 51: 263-73. PMID 3472723 DOI: 10.1101/Sqb.1986.051.01.032 
• 1986, Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature. 324: 163-6. PMID 3785382 DOI: 10.1038/324163A0 
• 1987, Mullis KB, Faloona FA. [21] Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction Methods in Enzymology. 155: 335-350. DOI: 10.1016/0076-6879(87)55023-6 

Articles: 

• The History of PCR
• Performance evaluation of thermal cyclers for PCR in a rapid cycling condition
• PCR – A critical examination
• PCR Cycling Parameters–Six Key Considerations for Success
• What is PCR? – The Beginner’s Guide
• CORMAN-DROSTEN REVIEW REPORT BY AN INTERNATIONAL CONSORTIUM OF SCIENTISTS IN LIFE SCIENCES (ICSLS)
• The many scandals of the PCR test: Part 1
• The many scandals of the PCR test: Part 2  

Videos:

• What is PCR? Polymerase Chain Reaction | miniPCR bio™
• Coronavirus Test: Real time RT-PCR – Animation video
• RT-PCR for diagnosing COVID-19 (former 2019-nCoV)