PhIP can accurately identify known and previously unreported autoantigens - Coursework Example

Analysis of the experimental procedures used in figure Analysis of the experimental procedures used in figure 3 The hypothesisof the experiment is “To prove that that PhIP can accurately identify known and previously unreported autoantigens “ Flow chart of experimental program of works The experimental procedures in figure 3 are shown in the flow chart above. The experiments seek to affirm the hypothesis that “PhIP can accurately identify known and previously unreported autoantigens” figure 3 shows seven figure that represent various experimental processes.

This summary shall try to analyze those experimental processes and the key technicalities behind them. Key technical issues in the program of work include the accurate and PhIP sequencing and T7 cloning, verifying and analyzing results from human samples with different consenting PND patients. Design of T7-Pep, (this is for explanation of fig 3.(c)) Firstly the human protein and cDNA sequences available are downloaded from the database at build 35.1 of the human genome. Then this cDNA and the proteins are used to construct the library.

Cloning of T7-Pep This procedure involved the proteome wide library and the N/C terminal libraries that were PCR amplified as 23 independent pools with common primer sequences. This process was conducted in a controlled thermal profile. The polymerase chain reaction was then digested and cloned into the T7FNS2 making numerous copies of each peptide.the cloning process involved the inserting a peptide in the required ORF sequence selected using restriction enzymes. Patient samples. This part is an a explanation of the processes involved in fig 3.

d, g, f, c, e Patient A, B and C samples are collected from willing patients approved by relevant medical authorities. The samples of cerebrospinal fluids were kept at temperatures of - 80 degrees Celsius before being used. Before the collection of sample the patients have to undergo various neurological evaluations before the samples are collected for confirmation. Dots blot validation of candidate autoantigens. (Explanation of fig 3 (a) (c) The complete collection Open reading frames of the genome was generated by the PCR and gateway recombination cloning as a source for testing antoantigen candidates by immunoblot.

Western blotting was used for immunoprecipitation as shown in fig 3(a). Western blot as fig 3(a) with cerebrospinal fluid (CSF) from all the patients; patient A,B and C was used to validated the candidate autoantigens. There was full staining for the full-length TGIF2LX –GFP that is usually expressed in 293T cells. Individual clones made have to be verified and tittered. In western blot proteins are usually separated by gel electrophoresis, nitrocellulose is the sheet of paper used for transfer of proteins.

The blot is usually incubated with a generic protein like milk to absorb to any sticky places on the nitrocellulose before an antibody is then added to the solutions which are protein specific. The antibodies are usually colored to enable observation. The location of autoantigens are then located by incubating it in a substrate that brings out the blot levels that are recorded and analyzed for comparison. The photographed films are then analyzed using computer software such as image J software Conclusion From the figure we can conclude that PhIP can accurately identify known and previously unreported autoantigens from the various experimental procedures.

References Srivastava, S. (n.d.). A two-parameter generalized Poisson model to improve the analysis of RNA-seq data. Retrieved November 3, 2011, from http://nar.oxfordjournals.org/content/38/17/e170.long