DNA Repair Study Usin

Studying DNA repair mechanisms often involves introducing specific lesions into plasmid DNA and analyzing repair efficiency by restriction enzyme digestion and gel electrophoresis. This article discusses a scenario where a 6.4 Kb plasmid with two restriction sites—HindIII and EcoRI—is modified by introducing a G:T mismatch at the HindIII site. After incubation in a repair system with 50% efficiency, the resulting digestion pattern on an agarose gel is predicted.

Background: Plasmid and Restriction Sites

• Plasmid size: 6.4 Kb

• Restriction sites: HindIII and EcoRI, yi

  • 3.1 Kb fragment

  • 3.3 Kb fragment

• elding two fragments upon double digestion:

Effect of G:T Mismatch at HindIII Site

• The HindIII recognition sequence is palindromic (5′-AAGCTT-3′).

• A G:T mismatch in one strand

•  of the HindIII site disrupts the recognition sequence.

• Restriction enzymes like HindIII are highly sequence-specific and generally do not cut if their recognition site is mutated or mismatched.

• EcoRI site remains intact and cuts normally.

Repair and Digestion Outcomes

• The plasmid is incubated in a repair system with 50% efficiency to correct the G:T mismatch back to the correct G:C base pair, restoring the HindIII site in half of the plasmid molecules.

• After repair, the sample is digested with both HindIII and EcoRI.

Expected Band Patterns on Agarose Gel

1. Unrepaired plasmids (50%):

   • HindIII site remains mutated (no cleavage at HindIII site).

   • EcoRI site is cleaved normally.

   • Result: One linear fr

   • agment of approximately 6.4 Kb (since only EcoRI cuts once, the plasmid remains circular or nicked but not cut into two fragments).

2. Repaired plasmids (50%):

   • HindIII site restored and cleaved normally.

   • EcoRI site cleaved normally.

   • Result: Two fragments of 3.1 Kb and 3.3 Kb.

Overall Gel Pattern

• Three bands expected:

  • One band at ~6.4 Kb (uncut or single-cut plasmid) from unrepaired plasmids.

  • Two bands at 3.1 Kb and 3.3 Kb fro

  • m completely digested, repaired plasmids.

• The intensity of the 6.4 Kb band and the sum of the 3.1 Kb and 3.3 Kb bands should be approximately equal, reflecting 50% repair efficiency.

Summary Table of Bands

Keywords for SEO Optimization

• Plasmid DNA repair assay

• HindIII and EcoRI digestion

• G:T mismatch repair

• DNA repair efficiency assay

• Restriction enzyme site mutation

• Agarose gel electrophoresis DNA fragments

• DNA mismatch repair system

• DNA repair and restriction digestion

• Molecular cloning and DNA repair

• DNA lesion correction assay

Conclusion

When a 6.4 Kb plasmid containing HindIII and EcoRI sites is damaged by a G:T mismatch at the HindIII site and repaired with 50% efficiency, digestion with both enzymes yields a gel pattern with:

• A 6.4 Kb band from unrepaired plasmids (HindIII site mutated, no cut).

• Two bands of 3.1 Kb and 3.3 Kb from repaired plasmids (both sites cut).

This pattern reflects the partia