What is the Difference Between Klenow and T4 DNA Polymerase?

06/25/2025 • Giovanni Medeiros

The main difference between Klenow and T4 DNA polymerase lies in their origins and temperature requirements. Here are the key differences:

Origin: Klenow fragment is a large fragment of E. coli DNA polymerase 1, while T4 DNA polymerase is the DNA polymerase 1 of bacteriophage T4.
Temperature: Klenow fragment can be used at 25°C or room temperature, but T4 DNA polymerase must be used at 12°C due to its robust exonuclease.

Both Klenow and T4 DNA polymerase are prokaryotic polymerases with 5′→3′ polymerase and 3′→5′ exonuclease activity. They lack 5′→3′ exonuclease activity, making them suitable for many applications in molecular biology. Both enzymes require a template and a primer to catalyze the synthesis of DNA and can be heat inactivated at 75°C.

Comparative Table: Klenow vs T4 DNA Polymerase

Klenow and T4 DNA polymerases are both used in molecular biology for DNA synthesis, but they have some differences. Here is a table comparing their characteristics:

Characteristic	Klenow Fragement	T4 DNA Polymerase
Origin	E. coli DNA Polymerase 1	Bacteriophage T4 DNA Polymerase 1
Structure	Large fragment of E. coli DNA Polymerase 1	Separate enzyme
5'→3' Polymerase Activity	Present	Present
3'→5' Exonuclease Activity	Present	Present
5'→3' Exonuclease Activity	Absent	Absent
Optimal Temperature	37°C	37°C
Error Rate	1 x 10^-6	1 x 10^-6
Blunt Ends	Yes	Yes

Both Klenow and T4 DNA polymerases have 5'→3' polymerase and 3'→5' exonuclease activity, but they lack 5'→3' exonuclease activity, making them more suitable for many applications in molecular biology. They are both used in various techniques, such as DNA sequencing, nick translation, and mutagenesis.