Kamila Balušíková

 DNA – sequence of genes, repetitive sequence of noncoding regions  RNA  Proteins gene expression

DNA of certain gene – all nuclear cells RNA of certain gene – only the cells, where this gene is expressed

 Monogenic and polygenic inherited diseases  Some types of tumors  Presence of infection (detection of pathogens)  Disease progress during the therapy  Identification of people in forensic medicine  HLA-typization in cases of transplantation …  Prevention - examination:- preimplantation - prenatal - presymptomatic

Variability in DNA sequence between different individuals of the same species  Allele polymorphisms physiological function, with frequency > 1% predisposition to polygenic diseases  Mutations pathological function, with frequency < 1% cause of monogenic diseases

Detection of certain polymorphism of the predisposition gene  TARGET ANALYSES  COMPLETE ANALYSES

 The localization and the whole sequence of a gene is known  The mutation of the gene is known An examination of family members is not needed

 The localization and the whole sequence of a gene is known  Mutations of the gene are unknown An examination of family members is necessary

DNA isolation PCR (amplification of a DNA region) + other analyses result visualization

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

 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 dyes (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

Separating of DNA fragments ( RNA, protein molecules ) according to their molecular weight (size) Principle: 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 (the larger the slower)

 Gel – sieve structure of polymer molecules with pores agarose x polyacrylamide › Different resolving power: polyacrylamide 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)

 Nucleic acid visualization in gel fluorescent dye is added to the gel (e.g. ethidium bromide) › Intercalates into the DNA structure › After light exposure, its complex excites photons (shines)

MW marker (bp = base pairs) DNA fragments

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) denaturation annealing extension

Basic compounds in PCR reaction  DNA sample  Pair of primers  Free nucleotides (dATP, dTTP, dCTP, dGTP)  DNA polymerase with buffer

 Short oligonucleotides (20 – 30 nucleotides)  Forward primer a reverse primer – one primer for one DNA strand  Complementarity 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 – depends on primers length and type of nucleotides

Sugar- phosphate skeleton base pairs bounded by hydrogen bounds

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) Temperature is a constant in each step

 Exponential function › Copies number of multiplying DNA region = 2 n, when n is number of cycles 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

 PCR with allele specific primers – target analyses (ASO-PCR = PCR with allele specific oligonucleotides)  PCR with general primers – followed by PCR product analysis – target, complete analyses

 Nested PCR (includes two successive PCR reaction)  Multiplex PCR (employs two or more PCR in same time – one reaction mix)

Unknown mutation – complete analyses  Sequencing searching for complete (exact) order of nucleotides in amplified 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)

 Gene expression levels – mRNA – proteins  mRNA – Real-Time PCR, Northern blot  Proteins – Western blot

Real-Time PCR → PCR for qualitative and quantitative analysis (x DNA diagnostic – qualitative analysis only) › We measure increasing amount of PCR product in time(how much?) in each cycle of PCR reaction › RNA cDNA (complementary DNA) Reverse transcription Reverse transcriptase

Real-Time PCR › 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 amplified → tested gen is more expressed than other gene or the same gene but under different conditions (comparative analysis)