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Why is DNA polymerase added at the end of PCR reaction?

Why is DNA polymerase added at the end of PCR reaction?



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PCR reaction is used to amplify DNA fragment. Each reaction requires a DNA template, buffer, dNTP mix and a unique pair of primers, one"forward"primer and one"reverse"primer. The DNA polymerase should be added last to the reaction, just before starting the PCR programme.

My question is, why is DNA polymerase added right before the PCR starts? Is it because it can begin to amplify DNA before adding the reaction to the thermocycler? How could it amplify the DNA when the DNA is not denaturated until added to the thermocycler?


True, the template is double stranded, but the primers could begin amplifying themselves, and any other stray DNA. You don't want any nonspecific amplification in the early stages that could compete with the target fragment. Also, even though Taq polymerase is a very hardy enzyme, it is standard practice to treat enzymes with special care, on ice so they don't degrade (although I have forgotten them on the bench overnight, and they survived that).


Why are nucleotides added to 3' end?

The DNA is only copied in the 5' to 3' direction because eukaryotic chromosomes have many origins for each chromosome in keeping with their much larger size. If some were copied in the other direction, mistakes will happen. It keeps every cell division on the same page, so to speak.

Because DNA synthesis can only occur in the 5' to 3' direction, a second DNA polymerase molecule is used to bind to the other template strand as the double helix opens. This molecule synthesizes discontinuous segments of polynucleotides, called Okazaki fragments. Another enzyme, called DNA ligase, is responsible for stitching these fragments together into what is called the lagging strand.

The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide, ("acceptor") with the 5' phosphate end of another ("donor"). The two "sticky ends" have to be in opposite directions for replication of the entire DNA molecule to be complete.

The average human chromosome contains an enormous number of nucleotide pairs that are copied at about 50 base pairs per second. Yet, the entire replication process takes only about an hour. This is because there are many replication origin sites on a eukaryotic chromosome. Therefore, replication can begin at some origins earlier than at others. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, forming two new molecules.


Why doesn’t PCR just use helicase and DNA Polymerase instead of using heat and TAQ polymerase to clone DNA Strands?

The technology is not mature enough to be a viable alternative to PCR for general lab use, but it has its advantages, one being that you don't need a thermocycler, but there are small, portable thermocyclers for small scale work.

What’s the benefit of HDA? I’ve never looked into the cost of a thermocycler, but I’d imagine it’d work out as cheaper than having to add a helicase to every run if you’re doing more than a few?

I've never heard of this. Does it just incubate at 55C or something?

You can do PCR this way, but I have no idea why you would. PCR is easy and has a very high success rate, unlike pretty much every other enzymatic reaction in the molecular biology toolkit. The reagents for this kind of PCR are also WAY more expensive than the reagents for a traditional PCR, assuming your lab has a thermocycler already (which most do for reasons beyond just running PCRs).

Enzymes can be really finicky. As a general rule, the more enzymes you have to use in a reaction, the more likely the reaction is to fail just because there are more finicky steps and more opportunity for something to go wrong.

Different enzymes also tend to be at their peak performance under different conditions (different temperatures, salinities, pHs, etc). Typically reactions that require multiple enzymatic steps will separate the reactions out into different tubes (first reaction in one tube, then transfer some of the product into a different tube with a different buffer for the next reaction, and so on). In a PCR, the helicase and polymerase would need to be functioning simultaneously, so this can't be done. When we run a reaction with two enzymes in the same tube, we have to optimize the conditions so both enzymes have the best performance possible. Typically though, they'll both be performing below 100% efficacy, which lowers the yield of the reaction overall. Even if the helicase and polymerase come from the same species originally and should theoretically work under the same conditions, the process of making the enzymes commercially useful can end up changing their properties.


PCR Procedure

Initialization

This step is necessary only for DNA polymerases that require hot-start PCR. The reaction is heated to between 94 and 96 °C and held for 1-9 minutes.

Denaturation

If the procedure does not require initialization, denaturation is the first step. The reaction is heated to 94-98 °C for 20-30 seconds. The DNA template’s hydrogen bonds are disrupted and single-stranded DNA molecules are created.

Annealing

The reaction temperature is lower to between 50 and 65 °C and held for 20-40 seconds. The primers anneal to the single-stranded DNA template. The temperature is extremely important during this step. If it’s too hot, the primer might not bind. If it’s too cold, the primer might bind imperfectly. A good bond is formed when the primer sequence closely matches the template sequence.

Extension/Elongation

The temperature during this step varies depending upon the type of polymerase. The DNA polymerase synthesizes a completely new DNA strand.

Final Elongation

This step is performed at 70-74 °C for 5-15 minutes after the final PCR cycle.

Final Hold

This step is optional. The temperature is kept at 4-15 °C and strops the reaction.


DNA and RNA both consist of nucleotides which contain a sugar, a base and a phosphate group. However there are a few differences. Firstly, DNA is composed of a double strand forming a helix whereas RNA is only composed of one strand. Also the sugar in DNA is deoxyribose whereas in RNA it is ribose. Finally, both DNA and RNA have the bases adenine, guanine and cytosine. However DNA also contains thymine which is replaced by uracil in RNA.

DNA transcription is the formation of an RNA strand which is complementary to the DNA strand. The first stage of transcription is the uncoiling of the DNA double helix. Then, the free RNA nucleotides start to form an RNA strand by using one of the DNA strands as a template. This is done through complementary base pairing, however in the RNA chain, the base thymine is replaced by uracil. RNA polymerase is the enzyme involved in the formation of the RNA strand and the uncoiling of the double helix. The RNA strand then elongates and then separates from the DNA template. The DNA strands then reform a double helix. The strand of RNA formed is called messenger RNA.


Molecular Biology Explained


A living organism must maintain itself continuously by producing the substances it needs to function. Proteins are one of these key substances and come in a seemingly infinite variety of shapes and structures. They are responsible for virtually everything that goes on in your body and cells, and are produced every second of an organism’s life. They are needed for various functions such as cell growth, tissue repair, signalling between cells, immune responses, muscle movement, enzymes to help carry out chemical reactions, hormones, and they also act as the building blocks for structures such as hair, feathers, insect shells and collagen.

The template for making proteins

Despite such huge variety, proteins are mostly defined by their sequence of amino acids linked together into ‘polypeptide chains’.

The template that codes for these proteins is a molecule called DNA – deoxyribonucleic acid.The central dogma- or more precisely hypothesis- of molecular biology states that genetic information translates from nucleic acid to protein, and not the other way round. This means that DNA makes RNA, and RNA makes proteins.There are exceptions to this process, such as when certain viruses are able to make DNA copies of their own RNA genome. But in most organisms, DNA is copied to RNA. The process by which DNA is copied to RNA is called transcription, and that by which RNA is used to produce proteins is called translation. This is the reason why DNA is such a key part of every organism’s vital processes.

Deoxyribonucleic Acid (DNA)

In humans, the nucleus of each cell contains 3 billion base pairs of DNA distributed over 23 pairs of chromosomes, and each cell has two copies of the genetic material. This is known collectively as the human genome. The human genome contains around 30 000 genes, each of which codes for one protein.

The deoxyribose nucleic acid molecule consists of a sugar molecule connected to one of four bases – guanine, cytosine, adenine and thymine. A Base joined to a sugar is called a nucleotide. Phosphate molecules join the ribose sugars together to form a chain , one side of the DNA double helix.


The four bases have different unique shapes which mean that they will only ever fit together in specific ways, like pieces of a puzzle. Adenine will only ever pair with Thymine, and Cytosine will only ever pair with Guanine. This means that the two strands of DNA are complementary, and effectively, are templates of each other.

Transcription

To see how DNA is used to make proteins, we need to introduce its sister compound Ribonucleic acid (RNA). RNA differs to DNA in a number of ways. The most important of these differences are firstly, that it is a single strand, rather than a double strand. And secondly, instead of Thymine, it has Uracil as a base.

Transcription is the process by which DNA is copied (transcribed) to messenger RNA (mRNA), which carries the information needed for protein synthesis.

So when a certain protein needs to be manufactured, the part of the DNA double helix which contains the relevant gene for that protein unzips, exposing the sequence of bases of A,C, G or Ts.

Then with the help of enzymes, free-floating nucleotides of mRNA link up with their corresponding nucleotides of DNA, and become joined together in a single strand of mRNA nucleotides.

Once the whole gene has become transcribed into a strand of mRNA, the mRNA moves off, and the DNA is zipped back up.

Translation

Now that the genetic code has been copied into mRNA, the code then needs to be translated to become a protein, and as mentioned earlier, all a protein really is, is a sequence of amino acids.

To do this, the mRNA is fed into a tiny molecular ‘machine’, called a Ribosome, which helps match up the correct amino acid for each part of the mRNA code. This is done for every three bases in the RNA sequence, called a codon. This three base sequence, gives a variety of different code combinations, each of which corresponds to a particular amino acid. So as the ribosome runs along the mRNA sequence, the correct amino acids are brought together in the correct sequence, and linked together to become a specific protein.

Our lab experiments

A lot of the experiments we run at the London BioHackspace are gene-typing experiments, which is essentially looking at our DNA and testing for the presence of certain genes.

For example, one of our projects is a test for certain blood types. We can do this because your blood group is determined by a certain combination of antigens in your red blood cells. Because antigens are proteins, coded in your DNA, we should be able to tell which antigens and which blood type you are, just by looking at your DNA.

DNA extraction

But first we need to get the DNA out. Not only is DNA locked in our cells, deep inside its nucleus, DNA is also tightly wrapped up with proteins. So we first need to take a collection of our own cells, normally by lightly rubbing the inside of our cheeks, and then we break open the collected cells, and extract the DNA using a number of chemical processes, which are outlined in the table below:

Polymerase Chain Reaction (PCR)

After breaking down the cells, and extracting the DNA, we now need to identify the gene we’re looking for. But there are a couple of problems. Firstly, we have now collected our entire genome of DNA, and not just the gene we are looking for. And secondly, it is such a small sample of DNA that we’re unlikely to be able to see anything at all. So we need to make lots of copies of the specific section of DNA we’re looking for, and we do this using a method called Polymerase Chain Reaction (PCR).

This is the exact same process that gets used with forensics, and is how they are able to match the DNA of a suspect, from just a small sample of hair.

To do PCR, we need to add a few ingredients to the tube, and then repeat a specific cycle of heating and cooling to get the DNA to replicate. This is done in a machine called a thermal cycler.


The thermal cycler will go through many cycles of heating the solution through different temperatures. The first stage involves heating to the hottest temperature, 96°C, which will cause the double DNA strands to separate. Then it will be cooled to allow the process of copying the DNA to begin.

In order to choose the gene we want to copy, we use primers. Primers are short pieces of DNA that are made in a laboratory. Since they’re custom built, primers can have any sequence of nucleotides you’d like.

In a PCR experiment, we use two primers which are designed to match the beginning and the end of the segment of DNA we want to copy. And in the case of our blood typing experiment, we’re using primers that match the beginning of the gene that codes for the blood antigens.

In the annealing stage, through complementary base pairing, one primer attaches to the top strand at one end of your segment of interest, and the other primer attaches to the bottom strand at the other end.

Then we heat the solution up to allow extension to begin. To copy the remaining sequence of DNA, we use an enzyme called DNA Polymerase. DNA Polymerase is a naturally-occurring enzyme whose function is to copy a cell’s DNA before it divides in two. When a DNA polymerase molecule bumps into a primer that’s base-paired with a longer piece of DNA, it attaches itself near the end of the primer and starts adding nucleotides.

Human DNA polymerase would break down at the temperatures we’re using in our PCR experiment. So the DNA polymerase that’s most often used in PCR comes from a strain of bacteria called Thermus aquaticus that live in the hot springs of Yellowstone National Park. It can survive near boiling temperatures and works quite well at 72°C.

In our PCR tube we’ve already added a mixture of four types of nucleotides found in DNA – A’s, C’s, G’s and T’s. So in the extension process, the DNA polymerase grabs nucleotides that are floating in the liquid around it and attaches them to the end of a primer, until the whole gene has been copied, and the end of a cycle is reached.

Then the process repeats all over again, with the two newly formed double strands of DNA themselves splitting, and being replicated. And so on… Until we have a highly concentrated solution of DNA for our blood type gene.

Gel electrophoresis

In the tube, even though we’ve successfully copied lots of fragments of DNA which are of the blood type gene, there are many other fragments left over from the PCR reaction. So now we want to be able to separate all of the various fragments of DNA in the tube, so that we can identify the blood typing gene fragment, and we do this by size.

To do this we use a process called Gel Electrophoresis. We make a gel out of a chemical called agarose, through which solutions of DNA are able to move, and we place our solutions of DNA from the PCR process into some wells which are indented in the gel. The gel also contains a chemical called Ethidium Bromide, which binds with the DNA, and acts as a fluorescent tag, which will allow us to see the DNA at the next stage.


The gel is placed in a solution and an electric current is passed through. Because DNA is a negatively charged molecule, as this current is applied, the strands of DNA will move slowly towards the positive electrode. However, the strands of DNA will move at different speeds. Shorter strands of DNA (for example short leftover chunks from the PCR reaction) find it easier to move through the gel, and so will move along much quicker. Whereas longer strands of DNA (e.g. gene sequences) will move more slowly through the gel. So after running the gel for a certain period of time, the different sized strands of DNA will be separated, appearing at different points along the gel.

/>But for us to find out exactly how long these DNA strands are, we need reference markers, which we call a ladder. This is a solution containing a mixture of DNA fragments of certain sizes (e.g. 300 nucleotides long, 500, 700, 1,000 etc.). When the ladder solution is placed into a well next to one containing our PCR DNA sample, it will run down the gel in the same way, with the various different sized chunks of DNA appearing at different positions down the gel. So if our PCR DNA sample, appears next to the ladder band representing 500 bp, we know our sample contains DNA of a gene roughly 500 nucleotides long. Or if it appears halfway between the bands of 1,500 and 2,000, we can make an estimate of it being roughly 1,750 nucleotides long.

Gel visualisation
Once the gel has run for long enough, we switch off the current, and then place the gel under UV light. The fluorescent marker provided by the Ethidium Bromide we put in at the start, means that the DNA will glow quite clearly under the UV.


As you can see, some clear bands of DNA appear roughly around the 347 base pair mark, which is the length of the DNA sequence we are looking for involved in determining blood group. Once we have this section, we add Alu1 restriction enzyme, which cuts the sequence if it is a blood group B allele, but not if it’s A or O.


Organisms have short sequences of bases which are repeated many times. These are called satellite DNA. These repeated sequences vary in length from person to person. The DNA is copied using PCRand then cut up into small fragments using restriction enzymes. Gel electrophoresis separates fragmented pieces of DNA according to their size and charge. This gives a pattern of bands on a gel which is unlikely to be the same for two individuals. This is called DNA profiling. DNA profiling can be used to determine paternity and also in forensic investigations to get evidence to be used in a court case for example.

For a suspect look for similarities between the DNA found at the crime scene and the suspect. For a paternity test, look for similarities between the child and the possible father.


Frequently Asked Questions

How accurate are PCR test results?

Rates vary by disease and collection method, but PCR test results are highly accurate, according to medical studies. They fare well on both measures of accuracy:

  • Sensitivity (the ability to identify even small amounts of a pathogen)
  • Specificity (being able to distinguish one pathogen from another)

What is multiplex PCR testing?

Multiplex PCR testing is when one test looks for multiple infectious agents simultaneously. Examples are STD PCR tests that look for up to nine pathogens.

What information is included in a PCR STD panel?

The results you get after a PCR STD panel, whether from a doctor or self-test kit, should include information about:

  • What viruses, bacteria, or parasites were tested for
  • Whether your results are positive (you have an infection) or negative (you don't have an infection) for each pathogen

They may also have numbers representing the severity of an infection. Home test-kit results may offer further information about what to do if you did test positive for anything.


Methods and Technology for Genetic Analysis

The Polymerase Chain Reaction, or PCR, is a basic method used in molecular biology to produce copies of a small target region of DNA in a sample. The basics to PCR were discussed previously here. The copies of DNA produced by PCR provide researchers with sufficient copies for other applications in research including automated Sanger sequencing. Although there is basic methodology to most PCR methods, each reaction is different and requires optimization, a process for adjusting variables and producing a single desired product. There are several factors to consider when optimizing PCR such as total copies of target DNA, primer concentration, MgCl2 and deoxynucleotides, or dNTPs. Some of these variables depend on the total volume of the PCR reaction because the final concentration of the components in PCR should be constant depending on whether the reaction is 25 ul, 50 ul or 100 ul. In this article we will focus on two variables, the number of copies of the target DNA and primer concentration.

The Template: Target DNA

Generating copies of a target DNA region using PCR applications is not as sensitive to the quality of the template DNA when compared to Sanger sequencing. However, it is still advisable to use a relatively pure DNA sample free from salts and other contaminants. Clean template DNA has a better probability to generate a clean PCR product. The final diluted sample of target DNA is better diluted in water rather than buffer because buffers can interfere with difficult PCR amplifications.

The most important aspect of the target DNA to consider is the total number of copies in the reaction available for amplification. The target DNA provides the initial template for the amplification of the first set of products amplified and continues to provide the template for the remaining cycles. As PCR products are generated, they also provide copies of the target DNA used as a template for amplification. This is what allows PCR to generate millions of copies of a target region. Therefore, it is important that sufficient copies of the original target DNA are present in the reaction. Too many copies of the original target can lead to generation of false products early in PCR that also act as template DNA. The template DNA isolated from bacteria may consist of only a 2 million-base genome whereas the human genome has 3 billion bases. Therefore, bacterial genomic DNA will have far more copies of the target in a 50 ng sample than human DNA. For bacterial DNA 10E5 copies will require only 300 picograms of DNA. For human DNA 10E5 will require over 300 nanograms of DNA, a one million fold difference.

PCR conditions generally recommend 10E4 to 10E5 copies of the target DNA in the reaction independent of the total volume. There is some flexibility in the copy number of the target sequence. However, more copies of the target DNA will reduce specificity of the PCR reaction and likely produce a greater number of false products. The total number of cycles for PCR should be reduced when higher concentrations of target DNA are in the reaction.

Concentration of the Primers

Primers are the determining factor of what region of the DNA will be amplified by PCR. The forward and reverse primer must have an exact base match with the beginning and end of the target region. Excessive primer concentration is perhaps one important factor that often causes generation of false products in a PCR. Too much primer reduces specificity and this will allow primers to anneal in regions of the template that are not the target region. The results of excessive primers are often seen in unclean Sanger sequencing results because false products can be sequenced along with the desired target. The amount of forward and reverse primer should be limited to reduce potential false priming. Excessive concentrations of forward and reverse primers can also cause formation of primer dimer when the primers anneal and amplify themselves independent of the target DNA.

Primer concentration is one variable dependent on the total volume of the PCR reaction in order that sufficient copies of the primer find the target annealing sites. A total concentration of 0.5 micro-Molar (uM) to 1 uM is generally sufficient to amplify most target regions, although a smaller concentration may also work in some applications. Typically our lab uses a final concentration of 0.8 uM for most PCR reactions. The final judgment on primer concentration will be viewed after products are electrophoresed on an agarose gel in order to show the number of products amplified.

We use a relatively simple calculation to dilute primers to a final concentration of 10 uM as shown starting with the primary primer concentration of 1 micro-grams (ug)/ micro-liter (ul). It requires that the molecular weight (MW) of the primer is known and should be provided along with the primer.

1 ug/ul *1umol/MW (ug) *10E6 ul/l = concentration umol/l which equals uM

A primer with the final concentration of 200 uM will be diluted by adding 1 ul of the primer to 19 ul of water for a final concentration of 10 uM. This is our working concentration for PCR. For a final concentration of 0.8 uM, 2 ul of the forward and reverse primer are added to a 25 ul reaction whereas 8 ul of each would be added to a 100 ul PCR reaction.

Primer concentration is one of the more important variables to consider when optimizing a PCR reaction. Concentrations greater than 1 uM could often lead to primers annealing along non-target regions and the generation of false products. Insufficient concentrations of either primer could result in little or no amplification.


Affiliations

Food Microbiology and Fermentation, Department of Food Science, University of Copenhagen, 1958, Frederiksberg C, Denmark

Łukasz Krych, Josué L. Castro-Mejía, Daniel N. Moesby, Morten B. Mikkelsen & Dennis S. Nielsen

GenXone S.A., 60-476, Poznań, Poland

Łukasz Krych & Maciej Sykulski

Computational Biology and Microbial Ecology, Department of Biological Sciences, Universidad de los Andes, Bogotá, Colombia

Chemometrics and Analytical Technology, Department of Food Science, University of Copenhagen, 1958, Frederiksberg C, Denmark

COPSAC, Copenhagen Prospective Studies on Asthma in Childhood, Herlev and Gentofte Hospital, University of Copenhagen, Copenhagen, Denmark