Difference Between Klenow and T4 DNA Polymerase

1. What is Klenow polymerase?
2. Why use Klenow fragment in DNA sequencing?
3. How does T4 DNA polymerase work?
4. Does DNA polymerase 1 need a primer?
5. What are Okazaki fragments?
6. How many types of deoxynucleotide triphosphates are used in Sanger sequencing?
7. What is DNA polymerase function?
8. What is 5 '- 3 exonuclease activity?
9. What does an exonuclease do?
10. What is the function of polynucleotide kinase?
11. What is exonuclease activity of DNA polymerase?

What is Klenow polymerase?

DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3'→ 5' exonuclease activity, but has lost 5'→ 3' exonuclease activity (1). Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini.

Why use Klenow fragment in DNA sequencing?

The Klenow fragment is extremely useful for research-based tasks such as: Synthesis of double-stranded DNA from single-stranded templates. Filling in receded 3' ends of DNA fragments to make 5' overhang blunt. Digesting away protruding 3' overhangs.

How does T4 DNA polymerase work?

T4 DNA Polymerase catalyzes the 5'→3' incorporation of nucleotides into double-stranded DNA using single-stranded and primed DNA as a template. It possesses strong 3'→5' exonuclease (proofreading) activity but does not exhibit 5'→3' exonuclease activity. ... These DNA products are often used in blunt cloning.

Does DNA polymerase 1 need a primer?

To initiate this reaction, DNA polymerases require a primer with a free 3′-hydroxyl group already base-paired to the template. They cannot start from scratch by adding nucleotides to a free single-stranded DNA template. RNA polymerase, in contrast, can initiate RNA synthesis without a primer (Section 28.1. 4).

What are Okazaki fragments?

Okazaki fragments are short sequences of DNA nucleotides (approximately 150 to 200 base pairs long in eukaryotes) which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication.

How many types of deoxynucleotide triphosphates are used in Sanger sequencing?

In the presence of the four deoxynucleotide triphosphates (dNTPs: A, G, C, and T), the polymerase extends the primer by adding the complementary dNTP to the template DNA strand.

What is DNA polymerase function?

DNA polymerase is an enzyme that synthesizes DNA molecules from deoxyribonucleotides, which are the building blocks of DNA. The enzymes play an essential role in DNA replication, usually working in pairs to produce two matching DNA stranges from a single DNA molecule.

What is 5 '- 3 exonuclease activity?

The 5'-3' exonuclease activity is the only active component of the N-terminus fragment of DNA Polymerase I. The main duty of the 5'-3' exonuclease activity is to remove the RNA primers at the 5' ends of newly synthesized DNA so that the polymerase activity can fill in the resulting gaps.

What does an exonuclease do?

Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs.

What is the function of polynucleotide kinase?

T4 Polynucleotide Kinase catalyzes the transfer of the γ-phosphate from ATP to the 5´-terminus of polynucleotides or to mononucleotides bearing a 5´-hydroxyl group. The enzyme may be used to phosphorylate RNA, DNA and synthetic oligonucleotides prior to subsequent manipulations such as ligation and cloning.

What is exonuclease activity of DNA polymerase?

DNA Polymerase I possesses a 3´→5´ exonuclease activity or "proofreading" function, which lowers the error rate during DNA replication, and also contains a 5´→3´ exonuclease activity, which enables the enzyme to replace nucleotides in the growing strand of DNA by nick translation.