10. Gene expression

Question 1

Genomics

Answer 1

Certain chromosomes prefer territories close to periphery of nucleus
Relatively little genome expression in this region
Genomic DNA is tightly condensed with the aid of (non-)histones (chromatin)
RNA in nucleolus and cytoplasm

Question 2

Trascriptomics

Answer 2

mRNA
RNA levels vary with type of cells, cell or tissue

Question 3

Microarrays

Answer 3

Expression of genes –> mRNA
Lable endogenous mRNA (no enzymes so cannot replicate this)
Convert mRNA into DNA (cDNA by reverse transcriptases)
Label cDNA while synthesize,, hybridize that to a carrier containing short DNA pieces specific for genes to be analyzed
Only 100% match hybridize
Dual color u-array
Measure gene expression
PCR

Question 4

Process for dual color u-array

Answer 4

Different samples (tumor vs normal)
Isolate mRNA
Make fluorescent labeled cDNA (target)
Both samples mixed and hybridized together to microarray (probe)
Relative intensity determined using confocal laser scanner
(genes are called biomarkers)

Question 5

Measure gene expression

Answer 5

Quantifying gene expression (comparing conditions)
Elucidation of molecular events associated with a given condition
Identification of molecular markers for pathological processes
Identification of potential drug targets in pharmacogenomics

Question 6

PCR

Answer 6

DNA polymerase makes complementary strand (5’ to 3’)
Denature DNA (94C) (from dsDNA to ssDNA)
Use gene-specific primers to start replication
Use DNA polymerase to replicate DNA
Repeat many times (2^x)
Too many times: saturation (PCR product is participating in reaction

Question 7

PCR advantages

Answer 7

Straightforward (quick)
Sequence of boundary regions must be known
Smarter enzyme mixtures larger fragments can be amplified faster
Used to type DNA markers in gene mapping projects or to screen human DNA for mutations and variations in microsatellite strength
Small amount of DNA necessary to start with (forensic or viral induced cancer)

Question 8

PCR disadvantages

Answer 8

Small amount of DNA necessary to start (easily contaminated)
Semi-quantitative because reaction saturates easily (limit number of cycles, put on gel, analyze) + solution is real-time PCR????

Question 9

Gapdh

Answer 9

normalization, stabel during experimental conditions: house keeping gene