June 26, 2022

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What factors should you consider before choosing a DNA polymerase?

What factors should you consider before choosing a DNA polymerase?

 


What factors should you consider before choosing a DNA polymerase?  The DNA polymerase family is divided into prokaryotic and eukaryotic DNA polymerases, which are classified according to sequence homology and crystal structure.

 

They are all related to three main functions:

5’–3′ polymerase: add new nucleotides to the 3’end of the DNA chain, the enzyme mainly determines the connection speed
5’–3′ Exonuclease: Remove the nucleotide at the 5’end of the molecule, physiologically, it is used to remove the ribonucleotide primer used to copy the backward strand of DNA
3’–5′ Exonuclease: Remove the nucleotides at the 3’end of the DNA molecule, which will determine the accuracy (or proofreading activity) of the enzyme


Group A polymerase:

It is a replicative or repairing DNA enzyme. Replicating polymerases (such as T7 DNA polymerase, mitochondrial polymerase gamma, Taq polymerase) match free nucleotides to the template DNA sequence, while repair polymerases (such as E. coli DNA polymerase I, Bst polymerase) correct Amplification of errors in base pairing. Replicase has a high reaction speed, but lacks proofreading activity.

They also have 5’to 3’exonuclease activity, which is particularly useful if fluorescent probes are used for amplification detection because it will hydrolyze the probes and emit fluorescence.

Modified repair enzymes of group A polymerases (for example, Klenow fragment of Escherichia coli) Large fragments of DNA polymerase I or Bst pol lacking 5’to 3’exonuclease activity are particularly suitable for special reactions due to their strand displacement activity .

Group B polymerase:

Including replicative polymerases (for example, Pfu polymerase, phi29 DNA polymerase, E. coli DNA polymerase II), which are involved in the biosynthesis of the leading and lagging DNA strands during cell replication. They are very accurate and can perform 3’–5′ proofreading of newly synthesized DNA, but at the cost of reaction speed.

Group C polymerase:

Including prokaryotic replication polymerases involved in bacterial genome replication (for example, Escherichia coli DNA polymerase III). They also have 3′-5′ proofreading activities.

Group D polymerase:

Including some eukaryotic replicative polymerases (such as Pyrococcus fierce DNA polymerase II), and is closely related to the B family in function.

X family polymerase:

Including eukaryotic enzymes (eg, polβ, polμ, polλ) that are mainly involved in repairing DNA after chemical, oxidative or radiation-dependent damage.

Group Y polymerase:

Including prokaryotic or eukaryotic enzymes with low fidelity for the complete DNA chain capable of replicating damaged DNA (for example, E. coli pol IV and pol V).

RT family polymerase:

Including eukaryotic telomerase and reverse transcriptase found in viruses, which use RNA as a template to synthesize DNA strands. When using RNA templates to generate DNA (reverse transcription), members of the RT family are required.

Most of the enzymes used in in vitro experiments belong to the A, B or RT family.

 

 

 

Factors to consider before choosing a DNA polymerase

  • Thermal stability: How long the DNA polymerase can withstand the high temperature required for specific PCR cycling conditions and still maintain its best condition
  • Sustained synthesis ability: how many nucleotides can be integrated by the DNA polymerase in the process of synthesizing new strands before dissociating
  • Speed ​​or elongation: How fast the DNA polymerase can synthesize new strands, usually related to the ability to continuously synthesize
  • Specificity: In addition to the desired target, it replaces the desired target, and how many non-specific or unwanted (background) targets are also amplified
  • Fidelity: Does the DNA polymerase have 3’to 5’exonuclease or proofreading activity so that it can correct errors by removing and replacing mismatched nucleotides
  • Stability: how well the enzyme binds to a specific sample type
  • Efficiency: how well the polymerase can overcome PCR inhibitors when processing crude samples, or how efficiently it can amplify GC- and AT-rich DNA

 

 

Important functions of DNA polymerase

  • 3’–>5′ Exonuclease activity: The polymerase can cleave the newly introduced nucleotide and correct errors (proofreading).
  • 5’–>3′ Exonuclease activity: The polymerase can cut nucleotides in the direction of polymerization, allowing it to perform DNA repair. When synthesizing complementary strands, this activity is also used to remove attached probes.
  • Strand displacement: The polymerase can replace downstream DNA encountered during the synthesis process. If the polymerase also has 5’–>3′ exonuclease activity, the replaced DNA will be destroyed. If not, keep it intact.
  • Tolerance: The polymerase can use a template containing uracil, or dUTP can be used in the polymerization process. Adding uracil is a common technique to prevent cross contamination.
  • Result end: The polymerase produces blunt ends or adenine overhangs. This is related to downstream applications (such as cloning).

 

 


What to do before choosing a DNA polymerase suitable for the application?

Before identifying DNA polymerase, you should ask youself:

  • Do I need to clone, use my product for expression or mutation analysis with a DNA polymerase that has proofreading activity?
  • Do I want to expand my long-term goals?
  • Am I trying to identify genotypes with high accuracy?
  • What costs do I need to consider?

 

After identifying the DNA polymerase, you should:

  • Ask colleagues if they used any of the options under consideration
  • Consult with the technical representative of the company that provides the DNA polymerase of your choice
  • See literature
  • If you want to replace DNA polymerases that have been successfully used in the past, compare these two enzymes with known effective targets and primers
  • Re-optimize cycle conditions and reaction settings if necessary

 

 

Common misunderstandings when using DNA polymerase

Some common mistakes are made when using DNA polymerase, and many of them are related to the notion that reagents can be used interchangeably. By creating many engineered polymerase variants to improve fidelity, specificity and other factors, it is likely that even Taq polymerases from different manufacturers may not be exactly the same. Therefore, it is extremely important to use a buffer system with a specific polymerase. Mixing and matching may not always work!

Here are some examples, highlighting some common causes of errors:

  • Response speed: B family members (such as Pfu) tend to be slower than A family members (such as Taq) because they have proofreading activities
  • Overhang: Enzymes lacking 3’–5′ exonuclease activity (such as Taq) will add extra adenine at the 3’end, which is especially suitable for rapid cloning strategies that do not involve restriction enzymes, while proofreading enzymes will not
  • Accuracy: Due to its proofreading activity, enzymes belonging to group B are less likely to make mistakes when amplifying DNA by about 100 times. However, the error rate of group A enzymes is not too high, so they can be used for short and rapid amplification
  • Template specificity: Enzymes belonging to the RT family cannot be used to amplify DNA, and enzymes belonging to the A and B families cannot be used to amplify RNA (unless they are redesigned).
  • Optimum reaction temperature: The polymerase comes from different sources, and the reaction temperature range is very wide. The enzymes (Taq, Pfu) used in classic PCR reactions are usually derived from archaea (because they need to withstand high temperatures) and will work similarly, but if Klenow fragments or Bst polymerases are used, they will not work in conventional PCR settings. It works because their optimal temperatures are 37°C and 65°C, respectively.

 

(source:internet, reference only)


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