science world

                          Random primer labelling (primer extension) Ø Gene probes, clone or TCR amplified and oligonucleotide probes can be random prime labelled with radioactive isotopes and non-radioactive labelled DIG Ø Random prime labelling of DNA fragment (double or single stranded DNA was developed by Fein Berg and Bolgesteinds an alternative to nicker translation to produce uniformly labelled probes. Ø Double stranded DNA is degenerated and aneled with random oligonucleotide primer. Ø The oligonucleotide primer too 5`―› 3` polymerase which synthesis labelled probe in presence of labelled nucleotide pressure. PCR DIG labelling: -         DIG DUTP is incorporated during PCR cycle into the DNA strands amplified from the DNA target. Photo biotin labelling: - Photo biotin labelling is a chemical reaction not an enzymatic one. ...

                                 Chromatin Immunoprecipitation (CHIP) Ø These are numerous protein that function by interacting directly with DNA, such chromatin protein or the factor that perform replication, repair and transcription Ø It is critical to map the dynamics of protein-DNA interaction in living cell in order to fully understand the complex function. Ø The powerful technique of chromatin immunoprecipitation (CHIP) was developed to capture such interaction. Ø CHIP allows to reaches to detect the presence of any protein of interest at a specific DNA sequence in vivo. Ø It allows the detection of specific protein-DNA interaction in vivo. Ø CHIP on CHIP or CHIP sequence allows mapping of all the protein-binding sites for a given protein across the entire genome. Ø This method depends on the use of an antibody to detect the protein intere...

                                    Inter-membrane structure of                                                         ATP synthase ATP synthase also sometimes known as complex V. generates the ATP molecule and uses the proton motive force to release ATP molecule into the matrix of mitochondria. The structure of ATP synthase is quite elaborate and consist of two major region the Fo region and F1 region. F1 region: - (catalytic unit) Ø This region lies in the matrix of the mitochondria and is made up of five types of polypeptide chains α, β, γ, ε, δ chain. Ø There α and there β chain to form a hexameric α 3 β 3 ring structure that lies be responsible for catalyzing  the synthesis of ATP Ø Th...

                                                                    DNA helicase assay Ø Helicase is the enzyme that harness chemical energy of ATP to separate the two parental DNA strand at replicating fork is called a helicase. Ø Many DNA helicase have been identified in E . coli cells Ø The problem is in finding which of these is involved in DNA replication. 1.     Rep helicase 2.     DNA helicase II 3.     DNA helicase III Ø Without a functional helicase, the fork cannot more as DNA synthesis must had immediately. Ø The DnaB product was known to be an ATPase and the DnaB   protein was found associated with the primers which makes primers for DNA replicatio...

                                                          HOT-Start  PCR Ø Hot start PCR is to use to minimize unwanted DNA product. Ø Major disadvantages is PCR production of unwanted DNA amplification. Ø Second step in addition to primer and tag polymerase. We add specific antibodies to back to polymerase from annealing. Ø When temperature raises from amplification 720 0 c the specific antibodies detaches from tag polymerase and amplification being with great specificity. Ø Modification of polymerase normally we add some antibody that will coat and being tag polymerase in normal room temperature is inactive them in room temperature antibody will properly function. So that tag polymerase can not initiate the polymerase chain reaction. Ø As we start increase temperature at very first of the PCR reaction the tempera...

                                                                                knockout mice Knock out mice is a mice in which particular gene e.g. [IL-2 gene] is replaced by muted version of the cell. The first knock out mice was created by Mario martin and Oliver in 1989 for this received Nobel prize in 2007 knockout mice are general used to study function of particular gene and to study immune deficiency disorder. Procedure to create knock out mice Part A: -Constructed of mutated gene Gene to be modified. Is selected and isolated. Offer this similar copies in engineered and constructed with few modifications generally marker genes are in sorted for modification. These are two types of marker gene. Neomycin resistance gene with confirm resistance to neomycin is inserted insid...

                                                                                                       Filter binding assay Ø The term filter is used in to describe a variety of different techniques in biochemistry, immunology, virology and molecular biology. Ø The molecular biology a filter binding assay is used to characterized DNA protein interaction. Ø This particular technique is also referred to as filter assay or a protein binding assay and it can be used to identify and characterised both DNA binding protein and the DNA sequence that can interact with these proteins. Ø Nitro cellular fi...

                                                     Phage display technique Ø Phage display is a molecular technique that allows the expression of foreign polypeptide or peptide on their surface of phage particle protein-protein, protein-peptide, protein-DNA interaction can be studied. Ø This method was described by George smith 1985 Ø In this technique, the DNA encoding the protein of interest is fused with gene encoding out of the proteins that forms a viral coat protein. Ø Genetic engineering technique are used to inert the foreign DNA fragments into a suitable phage coat protein gene. Ø Phage display involved the use of filamentous phage such as Fd, F1, M13 where the foreign gene was incorporated into gene specifying a minor coat protein. Ø Filamentous phage M...