The key difference between nick translation and end filling is that nick translation is a process that creates labelled DNA probes for various hybridization reactions while end filling is a technique that creates blunted fragments by adding nucleotides into single-stranded overhangs.
Nick translation and end filling are two techniques used in molecular biology. Nick translation is used for labelling probes for hybridization in order to detect specific nucleotide sequences. End filling is used to make blunted fragments that had sticky ends with single-stranded overhangs. Both techniques have great importance, and they are routinely performed in molecular research labs.
CONTENTS
1. Overview and Key Difference
2. What is Nick Translation
3. What is End Filling
4. Similarities Between Nick Translation and End Filling
5. Side by Side Comparison – Nick Translation vs End Filling in Tabular Form
6. Summary
What is Nick Translation?
Nick translation is an important technique used to prepare labelled probes for various molecular biological techniques such as blotting, in situ hybridization, fluorescent in situ hybridization, etc. It is an in vitro method of DNA labelling. DNA probes are used to identify specific DNA or RNA sequences. With the help of a labelled probe, specific fragments can be marked or visualized from a complex mixture of nucleic acid. Therefore labelled probes are prepared using various techniques. Nick translation is one such method which produces labelled probes with the help of DNase 1 and DNA polymerase 1 enzymes.
Figure 01: Nick Translation
Nick translation process starts with DNase 1 enzyme activity. DNase 1 introduces nicks to the phosphate backbone of double-stranded DNA by cleaving phosphodiester bonds between nucleotides. Once the nick is created, free 3′ OH group of the nucleotide will be produced, and DNA polymerase 1 enzyme will act on it. 5′ to 3′ exonuclease activity of DNA polymerase 1 removes nucleotides from the nick towards the 3′ direction of the DNA strand.
Simultaneously, the polymerase activity of DNA polymerase 1 enzyme works and adds nucleotides to replace the removed nucleotides. If the nucleotides are labelled, the replacement will occur by the labelled nucleotides, and it will mark the DNA for identification. Finally, this newly synthesized labelled DNA can be used as probes in various hybridization reactions.
What is End Filling?
End filling is a technique used in molecular biology to make blunted fragments. Restriction digestion produces fragments with overhangs. These fragments may not be compatible to ligate into plasmid vectors. Vectors are often blunted to allow non-compatible ends to be joined. Therefore, fragments with overhangs can be blunted by adding the nucleotides to the complementary strand using the overhang as the template for polymerization. This process is known as end filling.
DNA polymerases such as Klenow fragment of DNA Polymerase I and T4 DNA Polymerase catalyze the end filling. They add nucleotides to fill in (5′ → 3′) and chew back (3′ → 5′). Once the sticky ends are filled, they become blunt, and they are ready to ligate into a vector.
Furthermore, end filling can be used to label DNA molecules. However, it can only be used to label DNA molecules with sticky ends. In comparison to nick translation, end filling is a gentler method which rarely causes DNA breaks.
What are the Similarities Between Nick Translation and End Filling?
- Both nick translation and end filling are molecular biological techniques.
- They can be used to label probes.
- Both are in vitro
What is the Difference Between Nick Translation and End Filling?
Nick translation is a technique that creates labelled probes for hybridization. In contrast, end filling is a technique that creates blunt fragments for ligation into vectors. So, this is the key difference between nick translation and end filling. Furthermore, Nick translation requires the use of 5’to 3′ exonuclease activity while end filling does not require 5’to 3′ exonuclease activity.
The below infographic tabulates the differences between nick translation and end filling in more detail.
Summary – Nick Translation vs End Filling
Nick translation is a method that incorporates radiolabeled nucleotides into DNA. It synthesizes labelled probes based on the activities of DNase 1 and E. coli DNA polymerase 1 enzymes. End filling, on the other hand, is a technique that produces blunt fragments. When the fragments have sticky ends (single-stranded overhangs), it is necessary to make them blunted ends in order to ligate into vectors. End filling adds compatible nucleotides and creates blunt end fragments in order to make them compatible for ligation. Thus, this summarizes the difference between nick translation and end filling.
Reference:
“Blunting: NEB.” New England Biolabs: Reagents for the Life Sciences Industry, Available here.
“Nick Translation – An Overview”. Sciencedirect.Com, 2020, Available here.
Image Courtesy:
1. “FISH (Fluorescent In Situ Hybridization)”By MrMatze – Own work (CC BY-SA 3.0) via Commons Wikimedia