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DNA (gene mutations, paternity, organs compatibility for transplantations)
RNA Proteins
(gene expression)
Basic steps:
› Cell lysis → DNA release› Protein removal
ProteaseAdsorption or extraction
› DNA precipitation by ethanol → impurities removal
› DNA dissolution in water or buffer
DNA diagnostic
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
Spectrofotometryabsorption maximum
for nucleic acids 260 nmfor 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) – comparison of two light intensities
DNA diagnostic
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
Gel – sieve structure of polymer molecules with pores
agarose x polyacrylamid› Different resolving power:
polyacrylamid separates DNA fragments varying in single nucleotide in their lengthsagarose 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
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
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 Tanneal.= [4x(G+C) + 2x(A+T) - 5]
DNA diagnostic
1.Denaturationbreaking of H-bounds in DNA double strand; separated strands are created (T > 94°C)
2.Annealingprimers connection to separated DNA strands (Tanneal. = ?)
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
Exponential function› Copies number of multiplying DNA region = 2n,
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
DNA synthesis
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
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
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
Gene expression levels – mRNA– proteins
mRNA – Real-Time PCR, Northern blot Proteins – Western blotX DNA analysis – Southern blot
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