• Enzynomics overview in video
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  • Polymerase
  • Overview of PCR polymerases

    Principle and application of Polymerase Chain Reaction (PCR)

    A revolutionary method of DNA amplication called PCR (Polymerase Chain Reaction) was first developed in 1985 by Dr. Kary Banks Mullis, a biochemist in Cetus Biotechnology Company. The  PCR method requires repeated cycles of a 3-step procedure to amplify a specific DNA region from a very low amount of template DNA: (i) denaturation of the template DNA, (2) primer annealing, and (3) DNA synthesis by polymerase. PCR was difficult in early days because it needs to add polymerase after each PCR cycle. PCR machines were developed after the discovery of Taq polymerase which maintains its activity over 90. Patents for the PCR method using thermostable polymerases were sold for $300,000,000 to Heffman-LaLoche Company. The PCR method has opened a new avenue in most areas of biology and medicine including molecular biology, microbiology, medical science, and forensic science. The PCR method is widely used to analyze hereditary diseases and for diagnosis of bacteria or virus infection, It also allows us to find criminals from a drop of blood or a single hair.

     

    Parameters that affect PCR amplification efficiency

    An optimum condition of PCR can vary depending on each PCR reaction. Each parameter for PCR should be adjusted for most efficient amplification of the desired DNA fragments. Common parameters that affect efficiency of PCR reactions are as follows: reaction time, incubation temperature, amount and type of polymerases, amount of substrate DNA, dNTP, and Mg2+.

     

    PCR polymerases

    The optimal amount of PCR polymerase ranges from 1 to 5 units in a 100-μl reaction volume. In general, two and half units of polymerase give rise to satisfactory results. Amounts of non-specific amplified products usually increase with higher amounts of enzyme used. On the other hand, use of too small amount of enzyme results in insufficient amount of amplified PCR products. nTaq (Cat.# P025, P050) synthesizes 1 kb per min. Half-life of nTaq DNA Polymerase at 95 is 50 min. nPfu-Forte is more heat stable and retains over 95% activity after 60 min at 95.

     

    Mg2+

    The optimal concentration of Mg2+ is between 1.5 to 5 mM. Generally, the use of 1.5 mM Mg2+ gives rise to satisfactory results in the presence of 0.2 mM dNTP. The concentrations of Mg2+ used affect the activity of PCR polymerase, fidelity of DNA synthesized, and primer annealing. Since Mg2+ is required to activate dNTP by chelating dNTP in a stoichiometric manner, increased concentration of Mg2+ is required if elevated levels of dNTP are used or a high level of EDTA are present in a reaction mixture.

     

    dNTP

    Concentrations of dNTP (deoxyribonucleotides triphosphates) are optimal at 50-500 μM for PCR, but 200 μM dNTP is most commonly used. Equimolar concentrations of four different deoxyribonucleotides triphosphates are required for a high efficiency of PCR. Low concentrations of dNTP often result in increased specificity and high fidelity of PCR reactions. Mg2+ concentrations should be increased with increased concentrations of dNTP. The dNTPs in the reaction that is not incorporated into DNA are damaged at a certain rate during repeated cycles of PCR reaction. After 50 cycles of PCR reaction, approximately 50% of dNTP will remain intact in the reaction mixture.

     

    Template DNA

    Recommended amounts of template DNA are 0.1-30 ng of plasmid DNA or a DNA fragment, or 50-500 ng of genome DNA in a 100-μl reaction volume. Amplification of DNA can be easily obtained with 105-106 molecules of DNA and as few as 10-100 DNA molecules can be readily amplified by multiple rounds of PCR or nested PCR. Cautions should be taken with inhibitory substances which are often present in impure DNA and interfere with PCR reaction.

     

    Reaction temperatures

    Initial denaturation

    Heat template DNA to 94-95 for 2-3 min, which convert duplex template DNA into singlestranded DNA in the first step.

     

    Denaturation during PCR cycles

    To convert amplified duplex DNA into single-strand DNA during the PCR cycles, denaturation for 20-30 sec at 94-95 is usually sufficient. Longer denaturation period than this reduces the efficiency of PCR reaction due to gradual inactivation of the PCR polymerases.

     

    Annealing

    This step allows primers to anneal to the template single-stranded DNA. Annealing temperature depends on the Tm of a primer sequence. In general, annealing is carried out at the temperature 5 lower than the Tm of the primer. Annealing at either higher or lower temperature than this often results in failure of PCR or amplification of nonspecific PCR products.

     

    Elongation

    The Taq polymerase requires 1 min of elongation time for amplification of 1-kb DNA fragment., whereas the Pfu polymerase requires 2 min/bk DNA. High PCR efficiency can be obtained with Enzynomics nPfu-Forte (elongation time, 1 min/kb, Cat.# P410, P425) which contains a PCR enhancing factor.

     

    Number of PCR cycles

    For most purposes, 25-35 cycles are appropriate. 40-45 cycles are often used when the amount of template DNA is limiting. Increasing the number of PCR cycles may result in amplification of nonspecific DNA.

    In theory, each cycle of PCR reaction doubles the amount of amplified DNA product (2n, n=number of PCR cycle). However, the actual amount of DNA amplified is lower than calculated due to many reasons such as inefficient annealing, gradual decrease in dNTP available for amplification, and loss of polymerase activity, which collectively render the reaction to reach a plateau. This is called a “plateau effect."

     

    Primers

    Selection or design of primer sequences is a key factor to successful amplication of desired DNA fragment in PCR reaction. It is imperative to design a pair of primers so that they anneal to the specific sequence in the template DNA. If they have homology to other regions than the sequence of interest, it is likely that nonspecific DNA is amplified as well. Appropriate concentrations of primers range from 0.1 to 0.6 μM. Use of excess amount of primers frequently gives rise to either nonspecific DNA amplification or primer dimers that result from the two primers (see below). The recommended length of primers is 15-30 nucleotides. The primers of 30 nucleotides or longer can be used to amplify longer DNA or to solve specificity problem. For optimal PCR efficiency, G+C content should be within 40-60% and Tm of the two primers should be similar. The 3’-end of primers should not contain more than 3 consecutive G or C in order to avoid non-specific amplification and the 3’-end of the two primers should not be complementary to each other in order to prevent the formation of primer-dimers. The 3’ end of the primers should not contain ATrich sequence, which hampers stable annealing to template DNA.

  • Table. PCR Troubleshooting

    Trouble : No PCR products

    Cause

    Solution

    Template DNA that could not be amplified

    (e.g., Template DNA with a high GC content)

    Add DMSO (2-5%) and reduce the enzyme concentration to 0.5 unit.

    Use other organic solvents that reduce Tm.

    Problems with template DNA.

    Examine concentration, quality and purity of template DNA.

    Confirm that template DNA is not degraded by agarose gel analysis.

    Carry out test PCR with a pair of other primers that were successful.

    Prepare a new template DNA.

    Low concentrations of polymerase

    Increase the amount of enzyme concentration by 0.5 unit.

    If necessary, increase the amount of enzyme up to 5 units in 100-μl reaction.

    Low concentrations of MgCl2

    Increase MgCl2 concentration by 0.25 mM.

    Inadequate PCR cycles

    Reduce annealing temperature.

    Increase the number of PCR cycles.

    Check whether the final elongation step (72, 5 min) is carried out.

    Unsuitable primers

    Prepare a new pair of primers.

    Inadequate primer concentration.

    Use of equal concentration of two primers.

    Titrate primers for their optimal concentrations (from 0.1 to 0.6 μM).

    Primer quality and storage

    Confirm that the primers are not degraded.

    Store primers at -15 to -30.

    Formation of primer dimers

    Carry out hot-start PCR

    Divide the PCR mixture into two submixtures, each of which is inactive, and

    combine them together just before PCR reaction.

     

    Trouble : Multiple or smeared PCR products

    Cause

    Solution

    Low annealing temperature.

    Increase the annealing temperature based upon the length of primers and their

    nucleotide sequences.

    Low concentration of primers or

    inappropriate primers

    Check whether primers are optimally designed.

    Determine the optimal primer concentration by titration between 0.1 to 0.6 mM.

    Use the same concentrations of the two primers

    Carry out nested PCR*

    Template DNA that could not be amplified

    (e.g., Template DNA with a high GC content)

    Add DMSO (2-5%) and reduce the enzyme concentration to 0.5 unit.

    Use other organic solvents that reduce Tm.

    Problem with DNA template

    Use template DNA after serial dilution.

    * Nested PCR : A PCR that uses the amplified products obtained from primary PCR as template and

    another pair of primers that are designed to anneal within the amplified product.

     

  • Selection guide of Enzynomics PCR polymerases 


     

    Characteristics of Enzynomics PCR polymerases 


     

  • Restriction endonucleases
  • · 1X EzBuffer I

    10 mM Bis Tris Propane-HCl (pH 7.0 @25 °C), 10 mM MgCl2, 100 μg/ml BSA

     

    · 1X EzBuffer II 

    10 mM Tris-HCl (pH 7.9 @25 °C), 10 mM MgCl2, 50 mM NaCl, 100 μg/ml BSA

     

    · 1X EzBuffer III 

    50 mM Tris-HCl (pH 7.9 @25 °C), 10 mM MgCl2, 100 mM NaCl, 100 μg/ml BSA

     

    · 1X EzBuffer IV 

    20 mM Tris-acetate (pH 7.9 @25 °C), 10 mM magnesium acetate, 50 mM potassium acetate, 100 μg/ml BSA

     

    · 1X EzBuffer BamH I 

    10 mM Tris-HCl (pH 7.9 @ 25℃), 150 mM NaCl, 10 mM MgCl2, 100 μg/ml BSA

     

    · 1X EzBuffer EcoR I 

    100 mM Tris-HCl (pH 7.5 @ 25℃), 50 mM NaCl, 10 mM MgCl2, 0.025% Triton X-100

     

    · 1X EzBuffer Acc III 

    10 mM Tris-HCl (pH 8.5 @25℃), 100 mM NaCl, 10 mM MgCl2

     

    · 1X EzBuffer Bal I

    50 mM Tris-HCI (pH 8.2 @ 25℃), 5 mM MgCl2

     

    · 1X EzBuffer Dpn II

    50 mM Bis Tris-HCI (pH 6.0 @ 25℃), 10 mM MgCl2, 100 mM NaCl

     

    · 1X EzBuffer Cfr10 I 

    10 mM Tris-HCl (pH 8.5 @ 25), 3 mM MgSO4, 100 mM KCl, 0.02% Triton X-100

     

    · 1X EzDiluent A

    10 mM Tris-HCl (pH 7.4 @25℃), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 200 μg/μl BSA, 50% glycerol

     

    · 1X EzDiluent B

    10 mM Tris-HCl (pH 7.4 @25 ℃), 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 500 μg/μl BSA, 50% glycerol

     

    · 1X EzDiluent C

    10 mM Tris-HCl (pH 7.4 @25 ℃), 250 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.15% Triton X-100, 200 μg/μl BSA, 50% glycerol

     

     

     

  • Overview and history

     

    Experiments of O. Avery (1942) and A. Hershey (1952) demonstrated that DNA is the principle genetic material. DNA became an important subject of intensive research after its structure was determined by F. Crick and J. Watson (1953). Discovery of the restriction endonucleases allows researchers to cleave DNA at specific sites, a great advantage over physical or chemical cleavage that results in random fragmentation of DNA. Restriction endonucleases have become a new tool for DNA manipulation, laying a foundation for modern biology that led to the development of genetic engineering, molecular biology, and genomics.

     

     

     

    Restriction/modification phenomenon

     

    Analogous to the immune response in mammals, bacteria such as E. coli possess a defense system against infection of bacteriophages. S. Luria (1952) and G. Bertani (1953) discovered that efficiency of plating (EOP) of a bacteriophage could be markedly different, depending on the E. coli strains used. For example, EOP was close to 1 when bacteriophages prepared from type-B E. coli strain were infected to the same type-B strain. When they were used to infect type-K E. coli strain, however, EOP was greatly reduced to approximately 10-4. On the other hand, EOP was again close to 1 when bacteriophages from type-K E. coli infected the same type-K strain. However, infection of type-B strain with bacteriophages from type-K E. coli reduced EOP to approximately 10-4. This phenomenon was referred to as restriction, because multiplication of bacteriophages obtained from one strain of E. coli was restricted in the other E. coli strain.

     

    The bacteriophages originated from type-B strain could still infect type-K strain. With a low EOP (10-4), this was due to the fact that some bacteriophages from type-B avoided restriction and could infect the type-K strain. The genetic materials of the surviving ones were modified so that they were no longer restricted by the new host strain being infected. Thus, bacteriophages produced from type-K strain easily infect type-K strain, while their infection of type-B strain is much more difficult due to restriction/modification - a defense system of bacteria against bacteriophages.

     

     

     

    Discovery, purification, and application

     

    The early history of restriction endonucleases was written several decades ago primarily by three scientists Werner Arber, Hamilton Smith, and Daniel Nathans who earned a Nobel Prize in Physiology or Medicine in 1978.

     

    W. Arber and D. Dussoix (1962) proposed a model that restriction/modification phenomena depended on two types of enzyme activities. One is a methylase that transfers a methyl group to DNA, and the other is a restriction nuclease that cleaves unmethylated DNA. "Suppression of bacteriophage growth" occurs via the cleavage of unmethylated DNA (foreign bacteriophage DNA) by restriction enzymes present in bacteria. In contrast, methylation of DNA renders its own DNA resistant to cleavage by restriction nucleases. However, foreign bacteriophage DNA can get methylated with a low frequency before restriction enzymes act, leading to low EOP (10-4) in a new host cell (modification phenomenon). Complete modifications of phage DNA through several generations of cultivation in the new host strain lead to loss of its ability to survive in the original host strain.

     

    W. Arber and S. Linn (1968) demonstrated that restriction endonucleases from E. coli possessed the ability to degrade DNA, and M. Meselson and R. Yuan (1968) purified a type l restriction endonucleases for the first time. However, this enzyme cleaved DNA non-specifically to the disappointment of many biologists. In 1970, H. Smith and his colleagues finally purified a type ll restriction endonucleases (Hind II) from Haemophilus which cleaves at a specific recognition sequence. D. Nathan and K. Danna used restriction endonucleases to cleave SV40 DNA undergoing replication and analyzed its cleavage products, expanding the applications of restriction endonucleases. This accomplishment provided P. Berg with a revolutionary idea to create recombinant DNA for the first time in 1972. Following the advent of recombinant DNA technology, discovery of many more restriction endonucleases and their use have led to rapid growth of molecular biology and biotechnology.

     

    So far, approximately 3,000 restriction endonucleases have been found that recognize 230 different recognition sequences. Restriction endonucleases are found mostly in bacteria. However, their presence has been confirmed in viruses, archebacteria, and even in eukaryotes. Approximately 25% of bacteria contain at least one restriction endonuclease and some bacterial strains contain as many as seven different restriction endonucleases.

     

    Restriction/modification system

     

    Typically restriction endonucleases are found together with one or two modification enzymes (methylase or DNA-methyltransferase). Modification enzymes, like restriction endonucleases, recognize the same sequences. However, they protect DNA from cleavage by adding a methyl group to one base in the recognition sequence. Hence the modified sequences go unrecognized by restriction endonucleases. A methyl group sticks out to the major groove in the double helix, interrupting the access of the restriction endonucleases. After DNA replication, DNA exits in a hemimethylated state, in which only one strand is methylated. Hemimethylation also disables action of restriction endonucleases. This partnership of restriction and modification enzymes is referred to as the "restriction/modification system." In some bacteria, the two enzymes exist separately, whereas in the other bacteria they are present together as a complex enzyme or as distinct domains of a single polypeptide.

     

     

     

    Classification

     

    Restriction endonucleases have traditionally been categorized into three groups. This classification depends on the configurations of the constituent subunits, cleavage position, cleavage sequence specificity, and requirements of cofactor. However, it is difficult to group all restriction endonucleases into only three types due to the high degree of divergence in amino acid sequences in restriction endonucleases

     

     

     

    Type l

     

    Restriction and methyl transferase enzymes exist together in one complex. The recognition sequence and cleavage site are different. In other words, they recognize a specific sequence, but cleave DNA non-specifically approximately 1,000 base pairs away from the recognition sequence. In addition, ATP, AdoMet (S-adenosyl methionine, SAM), and Mg2+ are required for enzyme activity. 

     

    Although these enzymes provided a good model for studies of restriction phenomenon observed in

     

    bacteria, they are not suitable for molecular biology experiments.

     

    Examples : EcoK l, EcoB l, CfrA l, StyLT ll

     

     

     

    Type ll and Type ll subclasses

     

    Cleavage occurs within or near the recognition sequence. Thus, this type of enzyme is well suited to DNA analysis or cloning in laboratories. Type ll restriction endonucleases include enzymes of vast diversity and thus are divided into many subgroups. For example, amino acid sequences of Type ll restriction endonucleases vary greatly, indicating that convergent evolution occurred from many distinct ancestral proteins.

     

     

     

    Orthodox type ll

     

    This is the most common type of restriction endonucleases, which cleaves DNA within the recognition sequence. This type of restriction endonucleases exists separately from its cognate modifying enzyme. Most of the restriction endonucleases commercially available belong to this category. Since these enzymes bind DNA as homodimers, their recognition sites contain palindromic sequences of 4-8 base pairs, and they cleave both strands of DNA. As a result, a 5' overhang and a 3' overhang or a blunt ended structure can be produced. Cleavage requires Mg2+, and the sizes of these enzymes are small, typically with 200-350 amino acids.

     

    Examples : EcoR l (Cat.# R002), BamH l (Cat.# R003), Hind lll (Cat.# R008), Kpn l (Cat.# R014),

     

    Not l (Cat.# R001), Pst l (Cat.# R019), Sma l (Cat.# R015), Xho l (Cat.# R007)

     

     

     

    Type llS

     

    These enzymes exist separately from modification enzymes like the orthodox type II, enzymes,

     

    but they cleave DNA outside the recognition sequence. The recognition sequences are

     

    continuous and asymmetric. Even though they consist of a single polypeptide, their DNA

     

    binding and cleavage activities are present in a distinct domain. DNA binding of this type enzyme

     

    on its recognition sequence requires a single enzyme molecule, but cleavage of DNA requires

     

    interaction with the other enzyme molecule. For this reason, these enzymes and more active on

     

    DNA molecules that contain multiple recognition sites. Cleavage activity requires Mg2+, and the

     

    sizes of these enzymes are intermediate with about 400-650 amino acids.

     

    Examples : Fok l (Cat.# R088), Alw26 (Cat.# R066) l, Bbv l, Bsr l, Ear l, Hph l (Cat.# R053), Mbo l (Cat.# R050), SfaN l, Tth111 l (Cat.# R038)

     

     

     

    Type llE

     

    These enzymes react with two recognition sequences. One is cut by the enzyme, while the

     

    other bound to the enzyme functions as an allosteric effector. Marked site preference can be

     

    observed in this type of restriction endonucleases.

     

    Examples : Nae l (Cat.# R035)

     

     

     

    Type llF

     

    These enzymes react with two recognition sequences like the Type llE enzymes, but they cleave

     

    DNA at both sites.

     

    Examples : NgoM lV (Cat.# R095)

     

     

     

    Type llT

     

    These enzymes such as Bpu10 l and Bsl l exist as a complex enzyme containing two different

     

    subunits with restriction and modification activities respectively. Bpu10 l is a heterodimeric

     

    enzyme, in which the two subunits form a single active site that recognizes asymmetric

     

    sequences. Bsl l is a heterotetramer which recognizes palindromic sequences.

     

     

     

    Type llG

     

    This type of restriction endonucleases requires AdoMet for activity like Type ll B (see below;

     

    Type llB). Both restriction and modification activities reside in a single polypeptide.

     

    Examples: Eco57 l

     

     

     

    Type llM

     

    This type of enzyme recognizes and cleaves methylated DNA.

     

    Examples: Dpn l (Cat.# R054)

     

     

     

    Type llB

     

    This subclass can also be categorized into Type lV. These enzymes contain three distinct

     

    domains for restriction, modification, and recognition activity in a single polypeptide, and they are

     

    active in the form of heterodimer or heterotrimer. The recognition sequences can be symmetric

     

    (Bpl l) or asymmetric. They require Mg2+ and AdoMet for restriction and modification activities

     

    and have a larger structure with 850-1,250 amino acids. The recognition sequences can be

     

    continuous or discontinuous (two halves of the recognition sequence are separated). They cleave

     

    10-12 base pairs away from the recognition sequence and produce a 3’ overhang.

     

    Examples: Bcg l, Bpl l, Bsp24 l, Bae l, Cje l

     

     

     

      Type lll

     

      Similarly to the Type l restriction endonucleases, restriction and modification enzymes exist in one

     

      complex. ATP is necessary for the enzymes to move along DNA. Cleavage occurs approximately 25

     

      base pairs away from the recognition sequence. Complete digestion is almost never obtained. This

     

      is because cleavage of one recognition sequence needs one additional intact recognition sequence

     

      on the same DNA running in the opposite direction. Thus, this type of enzymes has no laboratory

     

      value and thus is not availablecommercially.

     

      Examples : EcoP l, Hinf lll, StyLT l

     

     

     

      Other

     

    Homing endonucleases

     

    These are DNA cleaving enzymes which contain an intron or intein. Intron is a region present in

     

    precursor RNAs that is removed by a process called splicing, and intein is a region in precursor

     

    protein that is removed during protein maturation. The prefix " l -" or "P l -" is used to indicate the

     

    presence of intron or intein, respectively, in the enzyme. These enzymes bind relatively longer

     

    sequences (12-40 base pairs) and cleave inside the recognition sequences. The recognition

     

    sequences are rare to find even within a very large genome because they are long.

     

    This enzyme can catalyze cleavage of the sequences with some base replacements.

     

    Examples : l -Ceul , PI-Psp l

     

     

     

    Nicking endonucleases

     

    Nicking endonucleases are genetically modified enzymes. Natural restriction endonucleases are

     

    genetically engineered to cleave only one strand of DNA double helix. This can be achieved by

     

    disabling dimerization, or suppressing the cleavage activity of one of the two monomers. It can

     

    be used to convert supercoiled DNA into open circular DNA.

     

    Examples : N-Alw l , N-BstNB l

     

     

     

         Nomenclature

     

         The name of restriction endonucleases is determined by the genus, species, and strain of a

     

         bacterium in which a restriction endonuclease is found, and finally the order of discovery. For

     

         example, EcoR l was discovered from Escherichia coli RY13; "E" (capital) from Escherichia (genus),

     

         “co” (lower case, first two letters) from coli (species), “R” (capital) for RY13 (strain), “ l ” (Roman

     

         numeral) for the enzyme which was discovered first.

     

     

     

    Table 1. Classification of type II restriction endonucleases

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