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Thursday, July 31, 2014

Yeast Two Hybrid System - For Protein - Protein Interaction Studies

Yeast Two Hybrid system uses a reporter gene to detect the interaction of pair of proteins inside the yeast cell nucleus. In the yeast Two Hybrid System, The interaction of target protein to the protein will bring together transcriptional activator, which then switches on the expression of reporter gene.

The Yeast Two Hybrid system makes use of modular nature of gene activator proteins. These proteins bind to the DNA and activate the transcription of the gene.

How the Yeast Two Hybrid System Works

Even though it looks complex, Yeast Two Hybrid System is relatively simple to use in labs to study protein-protein interaction. As the protein-protein interaction occurs inside the yeast cell nucleus, proteins from every part of the cell and from any organism can be studied using yeast two hybrid system.

This is how the Yeast Two Hybrid System works:

The DNA sequence that encodes target protein is fused with the DNA that encodes the DNA-binding domain of gene activator protein using recombinant DNA technology.

Two sets of proteins are used:

·         Bait and the Prey

Target protein is fused to a DNA-binding domain that localizes it to the regulatory region of a reporter gene as “bait.”

Specially designed protein in the cell nucleus (“prey”).

Bait can bind to the regulatory region of the reporter gene, which acts as a bait and can be used to fish out the protein that interact with the target protein inside yeast cell. The interaction of Bait and Prey will bring about the activation and expression / transcription of reporter gene. The reporter gene is the one which will help the yeast cell grow on selective medium.

Many potential binding partners can be prepared by ligating DNA encoding the activation domain of gene activator protein to a large mixture of DNA fragments from a cDNA library.

Cells that express this reporter are selected and grown, and the gene (or gene
fragment) encoding the prey protein is retrieved and identified through
nucleotide sequencing.
Through Two Hybrid System a protein linkage map has been generated for most of the 6,000 proteins in yeast and similar projects are underway to catalogue the protein interactions in C. elegans and Drosophila.

A similar technique called reverse two hybrid system, which can be used to detect mutation of chemical compounds that are able to disrupt specific protein interactions. In reverse two hybrid system the reporter gene can be replaced with a gene that kills the cells when the bait and prey proteins interact. Eliminating a particular molecular interaction can reveal something about the role of the participating proteins in the cell. In addition, compounds that selectively interrupt protein interactions can be medically useful: a drug that prevents a virus from binding to its receptor protein on human cells could help people to avoid infections.

Alberts, Molecular Biology of the Cell
Cooper, Molecular Biology of the Cell
The Yeast Two-hybrid System - Paul L. Bartel, Stanley Fields

Monday, July 28, 2014

DNA Sequencing - Traditional and Next gen Sequencing Methods

DNA sequencing is the process of finding out the exact order of nucleotide present in the DNA molecule. DNA sequencing has revolutionized medical and biological research. Before going into the details of DNA sequencing let us have some basics on the DNA molecule.

Central Dogma of Molecular Biology

Nucleic Acid - DNA / RNA Overview

Nucleic acids are first discovered in 1869 by Miescher. Found as a precipitate that formed when extracts from nuclei were treated with acid. Compound contained  C, N, O, and high amount of P.since it was an acid compound found in nuclei therefore named nucleic acid.

DNA / RNA are made up of nucleotides, these are the building blocks. Nucleotides composed of a Nitrogenous base, a pentose sugar and a phosphate group.

DNA has Adenine,Thymine,Guanine  and Cytosine, where as in RNA the nitrogenous bases found are Adenine,Guanine, Cytosine and Uracil.

The sugar present in DNA is deoxy ribose, where as in RNA it is D-ribose. Thats the reason DNA is called as 2-deoxy ribo nucleic acid.

A nucleotide comprises of a Pentose Sugar, Nitrogenous Base and a Phosphate Group.

The arrangement of Nucleotide polymer in DNA and RNA is as follows:

In DNA Adenine pairs with Thymine by a double bond and Guanine pairs with Cytosine by triple bonds.

DNA Sequencing: 
  • Maxam Gilbert Method of DNA sequencing
  • Chain Termination Method / Sanger's Sequencing Method
Maxam Gilbert Method of DNA sequencing

Sanger's Sequencing

Sanger's sequencing method was developed by Frederick Sanger and colleagues. This method is also know chain termination method. Sanger's sequencing method requires single stranded DNA template, Primer, Taq DNA polymerase, dNTPs and modified nucleotides namely ddNTPs (dideoxy nucleotide triphosphate). when a ddNTP is incorporated into a growing chain, the chain terminates. ddNTPs are usually flourescently / radiolabelled. By looking at the emission of light from the ddNTP sequence of the DNA can be deduced. the sequencing product will be of varying lengths which can be separated by Gel / capillary electrophoresis to read out the base sequence.

Next Gen Sequencing Methods:
  • Ion Torrent Sequencing


  • Pyrosequencing

  • SOLiD Sequencing

  • Illumina Sequencing Technology



Illumina Technical Resources
Applied Biosystem, Technical Resources
Other Internet Sources