genomics exam 3

QPCR (quantitative polymerase chain reaction)

• suitable for analysis of single SNP or small subset of SNPs

• only known SNPs can be detected

DNA SNP Microarrays

• suitable for analysis of small or large subsets of SNPs

• only known SNPs can be detected

• Hybridization of the DNA fragments into arrays with SNPs-specific probes attached to solid surface

• DNA SNP arrays detect large structural variations in the genomes (copy number variations)

Next generation sequencing

• suitable for analysis of any number of SNPs, from small subsets to all SNPs and other variations in human genome

• all known and previously unknown SNPs can be detected

• Sequencing of whole genome or a subset of pharmacogenomics-related genes

• Different from Sanger sequencing since it is sequencing by synthesis and multi-parallel sequencing

Quantitative PCR SNP assays

• amplification of DNA fragments using SNPs specific probes

TaqMan Assays

• A TaqMan assay is a method used during PCR to detect a specific piece of DNA by making it give off light.

• Steps

  • DNA Probe is labeled with Reporter and quencher dyes

  • when connected through DNA, quencher blockers reporter fluoresence

  • Taq polymerase with 5’-3’ exonuclease activity synthesizes new DNA and simulatenously cleaves the probe and releases the reporter dye

  • fluorescence of the reporter is detected

Illumina Sequencing

• most popular Next generation sequencing platform

• Steps

  • genomic DNA fragmentation and library construction

  • cluster generation

  • sequence by synthesis

  • assembly to reference genome and analysis of variations

Assessment of combination genes effects on drug metabolism

• combinatorial approach allows more accurate prediction of effects on genetic variation on drug activity and ADR

• greatly increase complexity of analysis (9 genotypes for 2 genes vs 3 genotypes for 1 gene)

• multiple gene interactions complicates analysis and provides significant limitations for pharmacogenomics- based predictions of drug activity and adverse effects

effects of environmental factors

• gene expression is regulated by lifestyle and environment

• smoking elevates expression of CYP2D6 and enchase metabolism of CYP2D6 dependent drugs

  • smoking effects the levels of fluvoxamine since it is metabolized CYP2D6

• People with similar genetic backgrounds may have significantly different levels of expression of pharmacogenomics-related genes in response to differences in environment or life style

transcriptome

the entire set of all messenger RNA molecules, including all splice isoforms, in one cell or a population of cells

proteome

• the entire complement of proteins, including the modifications made to a particular set of proteins, found in an organism over its entire lifecycle, or in a particular cell type at a particular time under defined environmental conditions

• analyzation of the proteome is difficult, the technology of proteomics is not as mature as genomics, due to the lack of amplification schemes

proteomics

a large-scale comprehensive study of a specific proteome including information on protein abundances, their variations and modifications, along with their interacting partners and networks, in order to understand cellular processes

post-translational modifications

• modifications that occur on a protein, catalyzed by enzymes, afters its translation by ribosomes is complete

• generally refers to the addition of a functional group covalently bonded to a protein or to proteolytic processing

• affects activity, stability and localization of the proteins

• single protein may undergo multiple modifications

metabolomics

the study of small molecule metabolites in cells, tissues, and organisms that are present in biofluids such as plasma and urine

pharmacometabolomics

a field which stems from metabolomics, the quantification and analysis of metabolites produced by the body it refers to the direct measurement of metabolites in an individual’s bodily fluids, in order to predict or evaluate the metabolism of pharmaceutical compounds, and to better understand the pharmacokinetic profile of a drug

microbiome

the ecological community of commensal, symbiotic and pathogenic microorganisms that literally share our body space

Digoxin

• inactivation is effected by bacterial genome variations, gut microbiome composition, diet

• digoxin positively regulates the CGR operon and induces the operon’s inactivation

  • arginine inhibits the operon

sulfasalazine

remains inactive until it reaches the distal gut, where azoreductases encoded by the gut microbiome cleave the N-N bond to release 5-ASA