Science slam Flashcards
Real-time (TaqMan)- based genotyping
qPCR: amplification an quantification of PCR products by using fluorescent dyes
TaqMan: uses fluorogenic probe to enable detection of specific PCR product (Taq polymerase cleaves quencher dye from reporter dye, increasing the fluorescent signal).
Used for SNP genotyping.
Overexpression systems
Bacterial, Yeast or Complex eukaryotes. Stable expression, promoter necessary, robustness and survival, reporter gene and selection marker. Transient or stable transfection Random or directed Integration. Validation by RT-PCR or Western Blot.
Ion torrent
NGS. Library prep (fragmentation, adaptor ligation) > emulsion PCR (oligos on beads) > loading on ion chip (one bead per well) > nucleotide binding causes pH change > detection of voltage change > signal processing
Drug metabolism
Liver. CYP enzymes > genetic variability. Phase I and II. De-/Toxification.
Parralel Artificial Membrane Permeability Assay (PAMPA)
In-vitro-assay: model of passive diffusion across artificial membrane. High throughput assay to predict oral absorption (ability of drug to pass through GI/BBB/skin).
Donor plate - artificial membrane - acceptor plate (detection: UV, HPLC, LC/MS)
Nanopore / MinION
NGS method.
Membrane containing nanopores. Ion flow generates current > disrupted by biopolymer passing pore > measured by electrode
In vitro drug report assays
Transporters: SLC, ABC
Transporter assays: substrate potential, inhibition potential
Uptake/permeability/inhibition assays.
Single base primer extension (SNaPshot)
Used to identify specific nucleotide (SNP).
Labeled primer hybridizes so that 3’ end is directly adjacent to base of interest. Pol extents primer by single base > id of base.
In vitro drug metabolism assay
Microsomes: vesicle like, contain metabolic ER enzymes (accessible but not fully human-like)
Liver microsomes: from human/animal donor
Human hepatocytes
Gene expression microarrays
RNA > cDNA > labeling with fluorochrome dyes > bind to complementary probes > identification by fluorescence
(two channel: comparison of two samples by using diff fluorophores)
CRISPR/Cas9
Based on bacterial antiviral defense system. Cas9 nuclease (recognizes PAM) complexed with a synthetic guide RNA (gRNA: crRNA+trcrRNA) > genome can be cut at a desired location, allowing existing genes to be removed/new ones added in vivo.
SNP microarrays
Illumina Infinium (fragmented DNA samples bind to silica beads on BeachChips > single base extension) Affymetrix
Tandem repeats
STRs: 2-6 bp repeat for 5-50 times (microsatellites)
VNTRs: repeat of longer nucleotide sequence, 500-10000 bp (minisatellites)
Fingerprinting: forensic/ paternity analyses
HPLC
Technique in analytical chemistry used to separate, identify, and quantify. Relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components.
Copy number variations
Large DNA segment that has been duplicated/lost. Mostly inherited, rarely somatic. Mostly benign but can predispose to disease.
Origin: NAHR, NHEJ/MMEJ, replication
