DNA Sequencing using the Maxam-Gilbert Method

Maxam-Gilbert Sequencing With this method of DNA sequencing instead of synthesizing DNA in vitro and stopping the synthesis reactions with chain terminators, this method starts with full-length, end labeled DNA and cleaves it with base specific reagents. So how this method works with guanine bases, but the same principle applies to all four bases; First we end-label a DNA fragment we want to sequence.

This can be 5’- or 3’-end labeling. Next, we modify one kind of base. Here we use dimethyl sulfate (DMS) to methylate guanines. (Actually, this reagent also methylates adenines, but not in a way that leads to DNA strand cleavage.) As in the chain termination method, we do not want to affect every guanine, or we will produce only tiny fragments that will not allow us to determine the DNA’s sequence. Therefore, we do the methylation under mild conditions that lead to an average of only one methylated guanine per DNA strand. Next, we use a reagent (piperidine) that does two things: it causes loss of the methylated base, then it breaks the DNA backbone at the site of the lost base (the apurinic site). In this case, the G in the middle of the sequence was methylated, so strand breakage occurred there, producing a labeled trimer.

In another DNA molecule, the first G could not be methylated, giving rise to a labeled phosphate (the base and sugar would be lost in the chemical cleavages). Finally, we electrophorese the products and detect them by autoradiography, just as in the chain-termination method. Of course, we need to run three other reactions that cleave at the other three bases. There are several ways of doing this. For example, we can weaken the glycoside bonds to both adenine and guanine with acid; then piperidine will cause depurination and strand breakage after both As and Gs. If we electrophorese this A + G reaction beside the G only reaction, we can obtain the As by comparison. Similarly, hydrazine opens both thymine and cytosine rings, and piperidine can then remove these bases and break the DNA strand at the resulting apyrumidine sites. In the presence of 1M NaCl, hydrazine is specific for cytosine only, so we can run this reaction next to the C+T reaction and obtain the Ts by comparison.

Choosing the right DNA polymerase for your PCR

Success of the polymerase chain reaction (PCR) largely depends on the choice of the appropriate DNA polymerase. DNA polymerase is one of the major components needed for setting up a PCR. For PCR, a thermo-stable DNA polymerase is essential, so that it can endure higher temperatures during the cycling conditions. Therefore, thermo-stable DNA polymerases serve as a key player in the current methods of DNA amplification and sequencing.

Based on their amino acid sequences, DNA polymerases are categorized into six families: A, B, C, D, X and Y. Thermophilic enzymes are found in all the six families. Same reaction is catalyzed by all DNA polymerases, that is, adding nucleotides to the 3′-end of the DNA primer to synthesize the new DNA strand complementary to the template DNA. The thermo-stable DNA polymerases synthesize DNA in a template-directed manner, and require primer-template hybrid to begin the synthesis.

Development of the PCR resulted in advancement of countless molecular biology techniques. Following PCR, a large number of various subsequent experimentation can be done. PCR for different purposes require different types of DNA polymerases. PCR is generally done for screening of the recombinant clone or for just simply determining the size of a particular DNA fragment. This can be carried out with simple DNA polymerases. But for cloning the DNA fragment for its expression, library construction, genome walking, RACE (Rapid Amplification of cDNA Ends) etc., different kinds of DNA polymerases are required.

Therefore, selecting an appropriate DNA polymerase, in accordance to the application, is extremely significant for the success of the experiment. DNA polymerases possess following four basic properties on the basis of which suitable DNA polymerase can be selected:

· Thermosatability

For the amplification procedures, DNA polymerases should be thermo-stable essentially. During each cycle of the PCR reaction, a denaturation step (at approximately 95°C) is there to separate two strands of the DNA molecule for next round of synthesis. Therefore, DNA polymerase should be durable enough to tolerate such a high temperature without losing its activity.

Half-life of a DNA polymerase at a specific temperature determines its thermostability. Its survival in a particular procedure largely depends on the factors like reaction mixture, protein concentrations and other reaction conditions.

· Extension rate

The number of dNTPs added per second per molecule of DNA polymerase is known as the extension rate. It highly depends on the reaction mixture and DNA templates. Sometimes there is formation of structures in the DNA molecules, as a result of which primer elongation ceases.

Generally, higher extension rates of DNA polymerases are desired. This facilitates amplification of longer DNA fragments.

· Processivity

Processivity is the probability that a DNA polymerase will not detach from the DNA after the attachment of a nucleotide, while translocating to the next position. It indicates the average number of the nucleotides that a DNA polymerase adds in a single binding event.

Similar to extension rate, processivity depends on the components of the reaction mixture and the sequence of the DNA template. On heterogeneous templates, processivity of each template position depends on the salt concentration.

· Fidelity

Fidelity denotes the frequency of insertion of the correct nucleotide per incorrect insertion. It actually refers to the ability of the DNA polymerases to insert correct nucleotides. It is an intrinsic property of the DNA polymerases. Therefore, DNA polymerases having low efficiency of correct nucleotide insertion, i.e. inefficient DNA polymerases, exhibit low fidelity, whereas, efficient polymerases exhibit high fidelity.

On the basis of the above mentioned properties, different types of DNA polymerases are available commercially. In accordance to the requirements and experiments, appropriate DNA polymerase can be selected from the following classes:

1) Taq DNA polymerase

Taq DNA polymerase was purified from bacterium Thermus aquaticus, found in hot springs. It is the most commonly used thermophilic DNA polymerase that catalyzes template-directed synthesis of DNA using nucleotide triphosphates.

It is used for general purpose PCRs, such as colony PCR (for screening of the recombinant clones), amplification of the DNA fragment for estimating its size, simple detection of the amplified product, PCR based molecular marker studies, etc.

They generally produce DNA fragments with ‘A’ overhang at 3’-end. This means that whenever a DNA fragment will be amplified with Taq DNA polymerase, the amplified products will have a single adenine base at 3’-terminal. Thus, the amplified DNA fragment can be directly cloned into T/A cloning vector.

Properties

· Taq polymerase possesses maximum catalytic activity at 75-80°C. It has half-life of 1.6 hours at 95°C.

· It has a extension rate of 1kb/minute.

· It has been observed that Taq polymerase dissociates from the DNA after attaching approximately 40 nucleotides,. Hence, exhibiting a good processivity.

· It lacks 3’ to 5’ exonuclease activity. As a result of this, it generally has higher error rate and less fidelity. It has error rate between 1X10^-4 to 2X10^-5 errors per base pair.

Limitations

Since Taq polymerases have higher error rate, it cannot be used for amplification of DNA fragments where high accuracy is desired. For example, it cannot be used for amplifying the DNA fragments to be cloned and expressed, and for mutagenesis studies.

2) Proofreading DNA polymerases

DNA polymerases are said to be proofreading when they possess 3’ to 5’ exonuclease activity. Whenever there in incorporation of non-complementary nucleotide in the growing DNA strand, the proofreading DNA polymerase by the virtue of its 3’ to 5’ exonuclease  activity, removes the erroneously attached bases by hydrolysis. This is an irreversible reaction. It significantly, increases the accuracy of the DNA synthesis from the template DNA, thereby exhibiting high fidelity.

Because of high fidelity, they are extremely useful for techniques that demand high accuracy in the DNA synthesis. For instance, for cloning and expression of amplified product, mutagenesis studies, etc. proofreading enzymes are obvious enzymes to choose.

They usually generate blunt-ended PCR products. Therefore, PCR fragments amplified by proofreading polymerases can be directly used for blunt-end cloning.

Properties

· Exhibits maximum catalytic activity at 68-75°C and have superior thermostability. Their half-life is 6.7 hours at 95°C.

· Relatively slower than Taq polymerase. Extension rate is approximately 0.5kb/minute.

· Their processivity is quite low as compared to Taq polymerase, i.e. approximately 4-30 nucleotides.

· Possess 3’ to 5’ exonuclease proofreading activity. They have far less errors as compared to Taqpolymerase. Their error rate is approximately 1.5 X 10^-6 error per base pair. This provides 5-15 fold higher fidelity than Taq polymerase.

Limitations

They are quite slower and therefore require more time to amplify the DNA fragments. Since they have low processivity, very high optimization is required. For this, generally gradient PCR is done.

Example

· Pfu DNA polymerase (Stratagene).

· Vent_R® DNA polymerase (New England Biolabs).

3) Polymerases for the amplification of long templates

Generally, Taq polymerase is able to amplify DNA fragment of ~3kb only. Whereas, the proofreading DNA polymerases can amplify PCR products of size up to ~6kb. Therefore, in order to amplify DNA fragments of much larger size, several DNA polymerases have been developed. Generally, they are mixture of a Taqpolymerase and a proofreading polymerase.

They are extremely useful for producing high yield of PCR product from genomic DNA with accuracy. They can amplify fragments as large as 30kb in size.

Properties

· Highly thermostable similar to Taq polymerase. Optimum temperature is usually 68°C.

· Extension rate is little bit higher than Taq polymerase, i.e. ~1.5kb/minute.

· Processivity similar to Taq polymerase.

· Due to inherent 3’ to 5’ exonuclease proofreading activity, they are 3-fold more accurate than Taqpolymerase.

Limitations

Since it is a mixture of Taq polymerase and proofreading DNA polymerase, its fidelity is not very high.

Examples

· Expand long template PCR system (Roche)

· LongAmp® Taq DNA polymerase (New England Biolabs).

4) Hot start polymerases

Hot start PCR is a modification of conventional PCR. It is used mainly to suppress non-specific product amplification and to increase the yield of the desired product.

In conventional PCR, the Taq polymerase remains active at room temperature and even on ice, to some extent. At this point, when all reaction components are mixed, primers can anneal non-specifically to the template DNA. The non-specific annealed primers can be extended by Taq polymerase, resulting in accumulation of non-specific products, thereby decreasing the yield of desired product.

Therefore, in hot start PCR, the DNA polymerase is kept inactive with the help of neutralizing monoclonal antibody until higher temperatures are reached. When the temperature raises, the antibody dissociates from the enzyme and gets inactivated making the DNA polymerase active, it considerably reduces non-specific priming, primer-dimer formation and, thus, increases the product yield.

They are very useful when the amount of DNA template is very low (i.e. less than 10⁴ copies of template DNA), when the DNA template exhibits high complexity (e.g. mammalian genomic DNA), and when several pairs of primers are there in the PCR (e.g. multiplex PCR). In all such cases, hot start PCR works best in combination with ‘touchdown PCR’ protocol.

Properties

· Remains inactive until higher temperatures are attained.

· Their extension rate and processivity is similar to that of Taq polymerase, since they are basically Taqpolymerase bound with an antibody.

· Lacks 3’ to 5’ exonuclease proofreading activity. Some suppliers provide blend of proofreading polymerase along with it to provide 3’ to 5’ exonuclease activity.

Limitations

Have lower fidelity and thus higher error rate.

Examples

· Advantage® 2 PCR enzyme system (Clontech).

· AccuStart^TM Taq DNA polymerase (Quanta Biosciences).

2) Next Generation Polymerases

Next generation of polymerases have been developed to overcome the shortcomings of the existing proofreading polymerases. They are engineered and generally are Pfu-based DNA polymerases. They are incorporated with several characteristics that provide them increased processivity, high fidelity and extreme speed.

By the use of high affinity DNA binding domain, their processivity has been increased dramatically. With the help of this domain, DNA polymerase can anchor much better. This prevents its early dissociation from the template DNA. Therefore, there is incorporation of more nucleotides per binding event due to improved processivity. This enhances PCR yield and shortens the extension time.

They are very suitable for high performance cloning in very short time. Their improved speed makes them desirable for higher throughput. They also provide very high yield of the PCR product. They are ideal for difficult targets and provide accurate results even with complex DNA templates having very high GC content (i.e. >80%).  They can amplify DNA fragments up to ~10kb. They are highly sensitive and can use very low amounts of DNA for amplification.

Properties

· Highly thermostable, as developed from Pfu polymerases.

· Modified to generate PCR fragments in much shorter extension times. With some next generation DNA polymerases, extension rate is as high as 1Kb/15-30second.

· Their processivity is 12-fold higher than the Pfu DNA polymerases.

· Highly accurate. Exhibits robust performance and reliability.

Limitations

They are relatively expensive.

Examples

· Herculase® II Fusion DNA polymerase (Stratagene).

· Phusion® DNA polymerase (Finnzymes).

Hence, from above discussion it is clear that for routine and general PCR you should use Taq DNA polymerase, whereas, if you have to express a gene or carry out mutagenesis experiment, you should go for the proofreading or new generation DNA polymerases. Similarly, if you need to carry out RACE or Genome Walking experiments, a Hotstart polymerase should be your pick. In addition, if you want to amplify large template then you should choose Expand Long DNA polymerase.

PCR Master Mix

The polymerase chain reaction (PCR) is used to amplify a specific fragment of DNA strand from a complex mixture of initial starting material. For setting up a PCR, you need to prepare a master mix. Generally, the PCR master mix consists of a target double-stranded DNA template, two oligonucleotide primers which hybridize to bordering sequences on either strands of the template, all four deoxyribonucleoside triphosphates (dNTPs) and a DNA polymerase.

Generally, you need to prepare 10-200µl reaction volume in small reaction tubes (200-500µl volume tubes) for setting up a PCR. The PCR is carried out in a thermal-cycler. Thermal-cycler is a machine that provides varying temperatures by heating or cooling the reaction tubes, as per the requirement of each reaction step. It is preferable if you use thin-walled reaction tubes, since it facilitates rapid thermal equilibration by maintaining proper thermal conductivity.

Now-a-days, in most of the thermal-cyclers, heated lids are there. As a result of this, the evaporating reaction mixture does not condense at the top of the reaction tube and remain within the reaction volume, maintaining the original composition. In thermal-cyclers without heated lids, a layer of mineral oil is added on the top of the reaction mixture to prevent evaporation. Alternatively, a wax ball can be inserted inside the reaction tube.

Once you have selected the appropriate target substrate, you need the following basic components for setting up the reaction

1)    Target DNA

You can obtain DNA for initial amplification from different types of sources. The template DNA source can be plasmid DNA, genomic DNA, cDNA, prokaryotic cells or even eukaryotic cells. You just need to simply boil prokaryotic or eukaryotic cell samples for extracting the DNA for PCR.

The minimal amount of template DNA required for PCR depends on the source. For example, 1µg of DNA is required if isolated from mammalian cells, whereas 1ng of DNA is sufficient enough if the source is bacteria. However, in case of plasmid DNA, you just need as little as 1pg of DNA for PCR amplification.

Double-stranded DNA (e.g. plasmid, genomic DNA etc.) as well as single-stranded DNA (e.g. cDNA) can be used as template DNA. Amplifications can be obtained both from circular-coiled DNA and linear DNA. However, it has been found that amplification from circular-coiled DNA is less efficient as compared to linear DNA. The main reason behind this is the huge and complex structure of circular-coiled DNA, which hinders in the proper binding of the primers to the template DNA, resulting in poor amplification. With most of the common PCR methods, DNA fragments of up to ~10kb can be easily amplified. However, using specialized techniques even the fragments up to 40kb can be amplified.

2)    Primers

The most crucial factor that decides the efficiency and specificity of the PCR amplification is the primers. Primers are the short stretches of oligonucleotides that anneals to the template DNA to amplify the fragment. They are essential for PCR amplifications because DNA polymerases can only bind the new nucleotides to the existing oligonucleotide/strand in 5’ to 3’ direction. Thus, primer hybridized to the template DNA serves as a short DNA strand to which the DNA polymerases can easily keep on adding new nucleotides to copy the existing DNA strand.

The primers are designed complementary to the DNA sequences bordering the target sequence to be amplified. But at the same time you must check that the forward and reverse primers are not complementary to each other, otherwise they will bind to each other to form primer dimers. This will obstruct proper DNA amplification, since no primer population will be left to anneal to the template DNA.

The other parameters that you need to keep in your mind while designing your primers are primer length, GC content and the melting temperatures. The ideal primer length is 20-28mer. The appropriate GC content is 40-60% and you should try to keep melting temperature in the range of 55-65ºC. There are many bioinformatics tools available with the help of which you can design your primers. Some examples are Oligo calculator, Generunner, Primer3, GeneFisher etc.

While designing primers the three objectives that you need to keep in your mind are:

i) The primers must be designed in such a way that there is high yield of the desired product.

ii) Amplification of unwanted non-specific sequences should be avoided.

iii) Subsequent manipulation of the amplified product shall be achieved easily.

3) dNTPs

Equimolar concentrations of dATP, dTTP, dCTP and dGTP must be present in the standard PCR. If you are using 1.5mM MgCl₂ along with the Taq DNA polymerase in 50µl reaction, then you should add 200-250µM of each dNTP. Higher concentrations of dNTPs should be avoided, since it reduces the yield by quenching the Mg²⁺ ions which are essential for proper activity of polymerases. Also, there will be more probability of incorporation of mismatched nucleotides by the polymerase.

Now-a-days, stocks of varying concentrations (10, 25 or 100mM) of dNTPs are available commercially. You should store stocks of these dNTPs at -20ºC. These stocks are provided with pH 8.1, which minimizes its damage from freeze and thaw. However, it would be preferable if you store these stocks in small aliquots, as this will prevent dNTPs from degradation by repeated freezing and thawing.

4) DNA polymerase

DNA polymerase is an enzyme needed for the synthesis of the DNA fragment. For PCR, a thermo-stable DNA polymerase is required, so that it can withstand higher temperatures. DNA polymerase needs a short stretch of oligonucleotides for the synthesis of new DNA fragment.

The polymerase gets attached to the primer-template hybrid and synthesis of new DNA strand begins using primers as a starting point. It keeps on adding single free nucleotides one by one to the exposed 3′-hydroxyl group provided by the primer. The DNA synthesis occurs in 5′ to 3′ direction only and not in 3′ to 5′ direction. This is because the nucleotides can be added only to the 3′ end and not to the 5′ end of the nucleic acid by DNA polymerase.

Now-a-days, a wide range of enzymes are available commercially. You can choose among them on the basis of their reliability, efficiency and ability to synthesize large fragments in accordance to your needs. IfTaq polymerase is used, then generally its 2-5 units are added in 50µl reaction volume. If higher amount of enzyme than this level is taken, then it may lead to accumulation of non-specifically amplified PCR product and there will be low yield of desired fragment. If accurate and flawless amplification of the DNA fragment is desired, then proofreading DNA polymerases are also available. For Example, Pfu DNA polymerase, Vent DNA polymerase, Phusion DNA Polymerase, etc. They remove mismatched nucleotides, if any, during amplification by their 3′ to 5′ exonuclease activity.

5) Standard Reaction Buffer

Buffer is the most basic component of the reaction master mix. It provides the platform for the reaction to take place. The main function of the buffer is to maintain the pH so as to make the reaction feasible. Any change in the PCR buffer will affect the consequence of the amplification. Basically, standard buffer consists of Tris-Cl at a concentration of 10mM.

During PCR cycling, when the temperature reaches 72ºC, there is a dip of more than a full unit in the pH of the reaction mixture. The optimal pH for the reaction mixture is ~7.2. Therefore, the pH of the standard reaction buffer is kept approximately 8.3-8.8, so that during the cycling process the pH of the reaction mixture may become desirable after getting reduced.

Buffer also contains KCl and MgCl₂ as the source of monovalent and divalent cations.

i) Mono-valent Cations

You need appropriate amount of mono-valent cations in the standard reaction buffer for primer annealing and for proper amplification of the DNA fragments. Generally, KCl is added as the source of mono-valent cations. Generally, 50mM KCl is sufficient enough to amplify larger DNA fragments. However, if the target DNA fragment is shorter, then higher concentration of KCl (70-100mM) would be suitable for the reaction.

ii) Di-valent Cations

Presence of free divalent cations plays a critical role in exhibiting proper activity by all thermo-stable DNA polymerases. You will find MgCl₂ as the most common source of divalent cations in most of the commercially available standard reaction buffers. Sometimes MnCl₂ is used alternatively but polymerases work less efficiently in this case. Generally, 1.5mM MgCl₂ is used in standard reaction buffers. Its higher concentration results in the production of non-specific amplified products, whereas inadequate amount reduces the yield.

Now-a-days, stock solutions of buffers are supplied along with the DNA polymerase. These buffers are optimized for particular type of DNA polymerase. For example, Taq polymerase is supplied with 10X standard Taq buffer. Similarly, different DNA polymerases are supplied with their respective buffers.

PCR : Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.

PCR principles and procedure

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. These components include:

• DNA template that contains the DNA region (target) to be amplified.
• Two primers, which are complementary to the DNA regions at the 5' (five prime) or 3' (three prime) ends of the DNA region.
• A DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.
• Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
• Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
• Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis.
• Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 20-150 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure

The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.

• Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.
• Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
• Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
• Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
• Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
• Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products

PCR stages

The PCR process can be divided into three stages:

• Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very specific and precise.
• Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
• Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

PCR optimization

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions. Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.

Application of PCR Isolation of genomic DNA

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs. PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.

Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing . This technique may also be used to determine evolutionary relationships among organisms.

Amplification and quantitation of DNA

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.

Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

PCR in diagnosis of diseases

PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.

PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques.

Variations on the basic PCR technique

• Allele-specific PCR: This diagnostic or cloning technique is used to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.
• Assembly PCR or Polymerase Cycling Assembly (PCA): Assembly PCR is the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.
• Asymmetric PCR: Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Melting temperature|Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.
• Helicase-dependent amplification: This technique is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.
• Hot-start PCR: This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
• Intersequence-specific (ISSR) PCR: a PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.
• Inverse PCR: a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.
• Ligation-mediated PCR: This method uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.
• Methylation-specific PCR (MSP): The MSP method was developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine,and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
• Miniprimer PCR: Miniprimer PCR uses a novel thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides, instead of the approximately 20 nucleotides required by Taq. This method permits PCR targeting smaller primer binding regions, and is particularly useful to amplify unknown, but conserved, DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. 16S rRNA miniprimer PCR was used to characterize a microbial mat community growing in an extreme environment, a hypersaline pond in Puerto Rico. In that study, deeply divergent sequences were discovered with high frequency and included representatives that defined two new division-level taxa, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity. By enlarging the "sequence space" that may be queried by PCR primers, this technique may enable novel PCR strategies that are not possible within the limits of primer design imposed by Taq and other commonly used enzymes.
• Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).
• Multiplex-PCR: The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
• Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
• Overlap-extension PCR: is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.
• Quantitative PCR (Q-PCR): is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR (see below), which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.
• RT-PCR: (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method, named RACE-PCR, short for Rapid Amplification of cDNA Ends.
• Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (the only primers present are covalently linked to solid support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR(where conventional Solid Phase PCR can be improved by employing high Tm solid support primer with application of a thermal 'step' to favour solid support priming).
• TAIL-PCR: Thermal asymmetric interlaced PCR is used to isolate unknown sequence flanking a known sequence. Within the known sequence TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.
• Touchdown PCR: a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5˚C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5˚C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.
• PAN-AC: This method uses isothermal conditions for amplification, and may be used in living cells.
• Universal Fast Walking: this method allows genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise') by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends) , 5'RACE LaNe and 3'RACE LaNe .

Five steps involved in the Expression and Purification of Proteins in E. coli

Production and detailed characterization of a variety of proteins is facilitated by their heterologous expression and purification. Recent advances in genomics have led to a massive increase in the number of proteins being produced using recombinant DNA technology.

In order to express heterologous proteins, a variety of expression systems have been developed. For example, bacteria (e.g. Escherichia coli, Bacillus subtilis, etc), yeasts (e.g. Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, etc), filamentous fungi (e.g. Aspergillus nidulance, Trichoderma reesei, etc), insects, plant cell cultures and mammalian cell lines.  

Generally, Escherichia coli is the most commonly used bacterial expression system for expression of the heterologous proteins. Reasons behind this include:

• Genetic manipulation is easy,
• Its culturing is inexpensive,
• Expression is fast,
• In many cases, the level of expression is high,
• Majority of foreign proteins are well tolerated, etc.

The above mentioned advantages and many more have guaranteed that E.coli remain a valuable organism for the high-level production of heterologous proteins.

In order to carryout expression of your gene of interest in E.coli, you should follow these steps:

Choosing the Expression vector

Choosing a suitable bacterial expression vector is the first step in the expression of heterologous protein in E. coli. The choice of vector system mainly depends on two factors. These include: i) Transcriptional and translational regulators, and ii) affinity tag.

i) Transcriptional and translational regulators

These elements consist of a set of genetic components that affect both the transcription as well as translation of the heterelogous protein. These elements include:

· Promoter

Promoter is the most important transcriptional element of an expression vector. Its main function is to allow RNA polymerase to bind to the DNA. Therefore, it regulates the rate of mRNA transcription. As a result of this, the amount of heterologous protein produced, largely depends on the type of promoter used.

The promoter which results in high level of transcription is called as strong promoter. However, weak promoter is the one which allows very low level of transcription. Consequently, an expression vector should have strong promoter to carry out highest level of transcription of the cloned gene. The commonly used strong promoters in bacterial expression vectors are, lac, trp, tac, and T7 promoters.

· Repressor

Repressors are the elements which allow the repression of a promoter. When a repressor is bound to the promoter no transcription occurs. Therefore, repressor works as a regulator of promoter activity.

It is very helpful if the protein of interest is toxic to the bacteria. By the use of repressor the accumulation of toxic level of a protein is monitored. Regulation of gene expression is achieved by the calculated use of the chemical.

· Terminator

The expression vector should have both transcriptional as well as translational terminators. A transcription terminator enhances the mRNA stability, therefore, leading to substantially increased level of protein production. In addition, it also stabilizes the plasmid by checking the expression of ROP protein, which is involved in the control of copy number of the plasmid.

Translational termination of the protein is carried out by the presence of a stop codon. Bacterial expression vectors contain stop codons in all the three open reading frames, in order to prevent ribosome skipping.

· Translational initiator

Translational initiation is determined mainly by the unique structural features at the 5’ end of the mRNA. This includes the ribosome binding site (RBS). Generally, the ribosome binding site consists of Shine-Dalgarno (SD) sequence followed by an AT rich translational spacer. These elements establish the translational efficiency of the mRNA.

ii) Affinity Tag

With the advancement of gene cloning methodologies, it is now possible to construct the fusion proteins. In this strategy, specific affinity tags are added to the protein sequence of interest. Addition of affinity tag to the protein of interest, simplifies the purification of target proteins by using affinity chromatography techniques.

Apart from this, use of affinity tags has the following advantages:

· It enhances the efficiency of translation of the target mRNA.

· It protects the target protein from proteolytic degradation.

· Some of the affinity tags (e.g. maltose binding protein or MBP) help in the solubilization of the target protein, hence target proteins remain in the cytoplasm rather than inclusion bodies.

The most commonly used affinity tags are:

His-tag which bind to the immobilized metal ions (e.g. Ni²⁺).

GST-tag which binds to the glutathione–Sepharose resin.

MBP-tag which binds to the amylose resin.

Cloning the gene of interest

After choosing the suitable expression vector, the next step is to clone your gene of interest. Majority of the expression vectors provide multiple cloning sites (MCS) for the ease of cloning of the gene of interest. Generally, the MCS is placed either between the RBS and the affinity tags or between the affinity tag and the terminator. Therefore, the cloning results in the fusion of affinity tag to N or C-terminal of the target protein, respectively.

However, care should be taken in checking out the frame of the fusion protein. For example, if you are doing a N-terminal fusion, then your sequence of interest should have stop codon. Similarly, if you are doing a C-terminal fusion, then you should maintain the open reading frame with the affinity tag and your sequence of interest should have the start codon (ATG).

After doing the transformation in E.coli, it is always better to confirm cloning by sequencing. You should compare the sequences in order to find out any frame shift mutation or a miss match. These anomalies can result in the incorporation of stop codons thereby leading to premature termination of your target protein.

Expressing the recombinant protein in E.coli

After confirmation of the cloning, the next step is to transform the recombinant plasmid into the E.coli host for protein expression. You cannot use the DH5? or similar strains for expressing your protein. For the expression of heterelogous proteins, certain protease deficient strains of E. coli have been developed. The most widely used strain is the BL21 (DE3).

Transform one vial of BL21 competent cells and select them on plates having the appropriate antibiotics. It is always good to prepare a glycerol stock of a single colony. If you are checking for the first time, then you should prepare stocks of 4-5 colonies separately. It is not a good practice to store the plate at 4°C and use it in future for expression studies. Either you prepare a stock from freshly transformed E.coli or do a fresh transformation. It is observed that these strategies improve the expression of your protein of interest.

For checking the expression you can start with a culture as small as 1ml. LB is the preferred media for the expression of a wide variety of proteins. You will have to optimize the growth conditions. Different proteins are expressed in different growth conditions. These include: temperature, aeration, size of the culture, culture media, concentration of inducer, etc.

Always remember to take a culture having blank vector along with your recombinant plasmid. This will work as a negative control. If you are using an inducible system, then you should also include the un-induced sample.

After doing all these stuffs, simply run the samples on SDS-PAGE along with the protein molecular weight marker. Meanwhile calculate the molecular weight of your recombinant protein (i.e. weight of your protein of interest + weight of the affinity tag). This will help you in examining the protein gel. If you got the band of desired size in the gel, congratulations! You have done it.

Unfortunately, if you are unable to find the band of desired size, check out the above written parameters or take another colony.

It is always good to check the solubility of the recombinant protein. Confirm that the protein is soluble by loading the clear lysate along with the cell pellet. If your protein is soluble then major fraction should be visible in the clear lysate as compared to the cell pellet.

Isolating the protein

After the confirmation of expression of your protein of interest in the soluble fraction the next step is to isolate the protein. For this, the cells are harvested through centrifugation. The next step is to disrupt the cells. Various methodologies have been developed for bacterial cell disruption. The most commonly used are treatment of cells with lysozyme followed by sonication.

Generally, the harvested cells are re-suspended in the lysis buffer. The lysis buffer contains lysozyme at a concentration of around 1mg/ml. Apart from this, certain protease inhibitors like PMSF are also included. The suspension is incubated at 4°C for about half an hour. Then it is gently agitated for nearly 10 min. After this, sonication is carried out. Care should be taken while sonicating the samples. Excessive sonication leads to the denaturing of the target protein.

After disrupting the cells, the suspension is centrifuged at low rpm for around half an hour to separate the lysate from the cell debris. The supernatant obtained after centrifugation is generally called as the cleared lysate. This lysate contains your protein of interest. Now you can proceed for the purification of the protein.

Purifying the protein

After getting the clear lysate the last step is to purify the protein, exploiting the affinity tag used. Depending on the fusion tag used, a suitable resin is chosen for the purification process.

The clear lysate is first allowed to bind to the resin. This is accomplished by mixing the clear lysate along with the resin and gently agitating them for around 30 min. After this the suspension is transferred to a polypropylene column. The suspension is allowed to settle down. After this, the cap of the column is opened and lysate is allowed to pass through the resin.

The next step involves the washing of the resin with appropriate wash buffer. Care should be taken in adjusting the stringency of washing. Higher stringency will lead to the loss of the protein of interest, whereas, lower stringency will cause purification of not target proteins. Therefore, stringency should be carefully adjusted in order to get large amount of target protein with no contamination.

After washing, the protein of interest is obtained by eluting it with the appropriate elution buffer. The composition of the elution buffer depends on the fusion tag used. The isolated protein can be stored at 4°C for shorter duration (around a week) otherwise it should be stored at -20 or -80°C.

This was just the overview of steps involved in the expression and purification of proteins in E.coli. We will be publishing the detailed reviews of each step in future articles. Please feel free to ask any questions by posting it in the comments.