Klenow Fragment (DNA Polymerase I Large Fragment)

High-Fidelity Polymerase with Optimized Exonuclease Profile for Precision Molecular Workflows

3′→5′ proofreading

No 5′→3′ exonuclease

Nuclease-free

DNase and RNase validated

Klenow Fragment (DNA Polymerase I Large Fragment), BR3P305 product image

Product overview

Klenow Fragment—also known as Klenow enzyme—is the C-terminal truncation derivative of E. coli DNA Polymerase I. Engineered to retain only the functional domains required for high-fidelity synthesis, it preserves the intrinsic 5′→3′ DNA polymerase activity and 3′→5′ exonuclease proofreading activity of the full-length enzyme, while completely abolishing the 5′→3′ exonuclease activity that causes non-specific degradation of template DNA. The retained 3′→5′ proofreading function minimizes misincorporation during DNA synthesis, delivering reliable high-fidelity performance for sensitive molecular applications.

Features and applications

Features

Optimized Exonuclease Profile: Eliminates unwanted 5′→3′ exonuclease activity to prevent template degradation, while retaining robust 3′→5′ proofreading activity for real-time error correction.
High-Fidelity Synthesis: The intrinsic 3′→5′ exonuclease activity removes misincorporated nucleotides during extension, reducing amplification bias and sequence errors for downstream assays.
Ultra-High Purity: Each batch is rigorously validated to be free of contaminating DNase, RNase, and non-specific nuclease activities, ensuring reproducible results for sensitive workflows.
Batch-to-Batch Consistency: Produced via a controlled recombinant expression system, delivering stable, predictable performance across all production lots.

Applications

NGS Library Preparation: End repair of fragmented DNA to convert cohesive ends to blunt ends; second-strand cDNA synthesis for RNA-seq workflows, where high fidelity minimizes sequencing errors.
Nucleic Acid Probe Labeling: Primer extension for radioactive, fluorescent, or biotin-labeled probe generation, enabled by the absence of 5′→3′ exonuclease activity that would degrade labeled primers.
Molecular Cloning: Blunt-end generation via 5′-overhang filling and 3′-overhang trimming, suitable for ligation-independent cloning and vector insertion.
Sanger Sequencing: Primer extension for dideoxy chain termination assays, where high fidelity ensures accurate base calling.

Components

Component	Specification/Notes
Klenow Fragment	10 U/μL
10×KF Buffer	10×

Performance data

Blunting of DNA 5′-Overhangs & Trimming of 3′-Overhangs

BR3P305 Klenow Fragment (DNA Polymerase I Large Fragment): blunting of DNA 5′-overhangs and trimming of 3′-overhangs

Compatible Applications: NGS library construction, vector ligation

Klenow Fragment (DNA Polymerase I Large Fragment) efficiently trims 3′-overhangs and fills 5′-overhangs of sticky-end DNA fragments, converting them into blunt-ended products. This reaction is mediated by the enzyme’s retained 3′→5′ exonuclease proofreading activity, with no risk of non-specific template degradation, preserving the integrity of nucleic acid termini.

Note: Blunt ends are the standard compatible substrate for NGS adapter ligation. This capability maximizes ligation efficiency, minimizes adapter dimer formation, and reduces ligation bias—a critical prerequisite for high-quality library preparation. The enzyme’s inherent proofreading activity further ensures accurate end modification for reliable downstream results.

Second-Strand cDNA Synthesis for NGS RNA Library Preparation

Compatible Applications: NGS RNA-seq library construction

The Klenow Fragment is optimized for second-strand cDNA synthesis in RNA library workflows: RNA is first reverse-transcribed into first-strand cDNA by reverse transcriptase. RNase H then degrades the RNA component of RNA-DNA hybrids, cleaving full-length mRNA into short oligonucleotide fragments that remain annealed to the first-strand cDNA. These RNA fragments serve as primers for Klenow Fragment-catalyzed second-strand cDNA synthesis.

Note: This workflow is a core step in RNA-seq library preparation. The Klenow Fragment’s 3′→5′ exonuclease proofreading activity ensures high-fidelity second-strand synthesis, minimizing amplification-induced errors and improving the accuracy of downstream transcriptome analysis—particularly for the detection of low-abundance transcripts.