Searching for microbes Part IX. Detection of nucleic acid. Ondřej Zahradníček To practical of VLLM0421c [email protected] Content of this slideshow. Introduction – Tale. Methods of nucleic acid detection. PCR principle. PCR practically. Check-up questions. Tale.
Introduction – Tale
Methods of nucleic acid detection
A DNA microarray (synonyms: gene chip, DNA chip, biochip) is a collection of microscopic DNA spots attached to a solid surface (glass, silicon).
Use: measuring ofexpression levels of large numbers of genes (104, even 106) simultaneously or to genotype multiple regions of a genome.
Each DNA spot contains only several molecules of a specific DNA sequence (oligonucleotide), known as probes (or reporters). The sample comes to the contact with the spot.
Now, molecules in the sample (called target molecules) hybridize with complementary molecules of the surface.
Hybridization is detected and quantified by fluorophore-labeled (or differently labeled) targets.
Results are almost processed by bioinformatic methods.
RNA chips and protein chips are simillar.DNA mikroarray („biochip")
Amplification of specific sections of DNA using PCR method
PCR development was enabled by research leading to finding Taq polymerase from a thermophilic bacterium Thermus aquaticus, being able to survive high temperatures.
The following is valid, not regarding the way of detection (gel electroforesis of ELISA)
Patients 1 and 4 – positive, patient 2 – negative, patient 3 – inhibition of reaction. 5 – positive control, 6 – negative control, 7 – ladder
Proper reaction line
Internal control line
The formulations may be something different!