Workshop 5

Question 1

What are the factors affecting the kinetics of hybridization ?

Answer 1

Temperature : Tm = 25 ̊C
Salt concentration : Rate increases with increasing salt
Base mismatches : Mismatches reduce rate
Base composition : Rate increases with increasing G+C
Fragments length : Probes shorter than target increase rate
Complexity of nucleic acid : Inversely proportional
Formamide/Urea : 20% reduces rate (higher conc. no effect)
Dextran sulfate : increases rate
Ionic strength : increased ionic strength increases rate
Viscosity : increased viscosity decreases rate of association

Question 2

How do microarray technologies work ?

Answer 2

• Microarray technology utilizes nucleic acid hybridization techniques
• Applications e.g. gene expression profiling, comparative genomic hybridization, SNP detection, alternative splicing etc.
• Thousands of gene can be analyzed in parallel.
• Gene chip arrays (Affymetrix) are fabricated using photolithographic technology to synthesis oligonucleotides directly on a glass surface.
• Hybridization to Affymetrix chips is DNA:RNA.
• Samples for hybridization are antisense copy RNA (cRNA) made in vitro using T7 RNA polymerase in the presence of biotinylated ribonucleotides bio-CTP and bio-UTP.
• After hybridization of the biotinylated cRNA, the chip is stained with streptavidin-phycoerythrin and read with a confocal scanner.
• Control and experimental samples are hybridized to separate chips. Comparison of the two chips is performed to determine the differential gene expression levels of the two compared samples.