2.  DNA (gene mutations, paternity, organs compatibility for transplantations)  RNA  Proteins (gene expression)

3. Basic steps: › Cell lysis → DNA release › Protein removal  Protease  Adsorption or extraction › DNA precipitation by ethanol → impurities removal › DNA dissolution in water or buffer DNA diagnostic

4.  Fenol-chloroform extraction (different solubility conditions in solvents)  Solving-out method (protein precipitation by NaCl)  Protein denaturation by heating  Adsorption method (silica-gel membrane) DNA diagnostic

5.  Spectrofotometry absorption maximum for nucleic acids 260 nm for proteins 280 nm → DNA concentration: at 260 nm → DNA purity is calculated by ratio 260/280 nm  Gel electrophoresis with fluorescent colors (approximate) › DNA is stained by intercalating dyes in gel › Gel is loaded with DNA standard (its concentration is pre-evaluated) – c omparison of two light intensities DNA diagnostic

6. Separating of DNA fragments ( RNA, protein molecules ) according to their molecular weight (size) on the principle of the movement of charged molecules in electric field  the nucleic acids consist of negatively charged phosphate groups → the movement direction goes from cathode (-) to anode (+)  The movement rate of DNA in gel depends on DNA fragment size in indirect proportion DNA diagnostic

7.  Gel – sieve structure of polymer molecules with pores agarose x polyacrylamid › Different resolving power: polyacrylamid separates DNA fragments varying in single nucleotide in their lengths agarose separates fragments which lengths differ minimally in 10 nucleotides (wider range – hundreds base pairs)  Etidium bromide – fluorescent dye which is added to the gel › Intercalates into the DNA structure › After UV exposure, its complex excites photons (shines) DNA diagnostic

8. The length of unknown fragments is compared to the length of standard fragments

9. PRINCIPLE : multiplying (amplification) of selected DNA part(s) Reaction is performed in cycles (30 – 40 cycles) Each cycle consist of 3 steps (change of temperature is constant affects individual steps) Basic compounds in PCR reaction  DNA sample  Pair of primers  Free nucleotides (dATP, dTTP, dCTP, dGTP)  DNA polymerase with buffer DNA diagnostic

10.  Short oligonucleotides (20 – 30 nucleotides)  Forward primer a reverse primer – one primer for one DNA strand  Are complementary to the sequences at the 3´end of corresponding DNA strand  Delimit the target DNA region which will be amplified  Their binding is influenced by temperature annealing teperature – depends on primers length and type of nucleotides T anneal. = [4x(G+C) + 2x(A+T) - 5] DNA diagnostic

11. Sugar- phosphate skeleton DNA diagnostic base pairs bounded by hydrogen bounds

12. 1. Denaturation breaking of H-bounds in DNA double strand; separated strands are created (T > 94°C) 2. Annealing primers connection to separated DNA strands (T anneal. = ?) 3. Extension (elongation) new DNA strand synthesis; DNA polymerase synthesize new DNA strand according to the old (template) one (T = 72°C) DNA diagnostic Temperature is a constant in each step

13. DNA diagnostic

14.  Exponential function › Copies number of multiplying DNA region = 2 n, when n is number of cycles DNA diagnostic first cycle (creating of two double stranded DNA molecules) second cycle (creating of four double stranded DNA molecules) third cycle (creating of eight double stranded DNA molecules) DNA synthesis Separation of DNA strands and primer pairing Separation of DNA strands and primer pairing Separation of DNA strands and primer pairing Target region of double stranded chromosomal DNA we want to amplify

15. ladder PCR fragments DNA diagnostic

16.  Nested PCR (includes two successive PCR reaction) – target analyses  Multiplex PCR (employs two or more PCR in same time – one reaction mix) – target analyses  PCR with sequence specific primers – target analyses (ASO-PCR = PCR with allele specific oligonucleotides)  PCR with general primers – followed by PCR product analysis DNA diagnostic

17. Unknown mutation – complete analyses  Sequencing searching for complete (exact) order of nucleotides in amplificated DNA fragment Known mutation – target analyses  Hybridization analysis of PCR product using labeled probe  RFLP (restriction fragment-length polymorphism) PCR product is specifically digested using restriction enzymes (restriction endonuclease – restrictase) DNA diagnostic

18.  Gene expression levels – mRNA – proteins  mRNA – Real-Time PCR, Northern blot  Proteins – Western blot X  DNA analysis – Southern blot

19. Real-Time PCR → PCR for qualitative and quantitative analysis (x DNA diagnostic – qualitative analysis only) › RNA cDNA (complementary DNA) › We measure increasing amount of PCR product in time(how much?) – in each cycle of PCR reaction › When target gene is not expressed, mRNA is not created – no amplification › The more of target gene mRNA, the more of cDNA, the faster is cDNA amplificated → gen is more expressed than other (comparative analysis ) Reverse transcription Reverse transcriptase RNA diagnostic