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

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Transcript of DNA (gene mutations, paternity, organs compatibility for transplantations) RNA Proteins (gene...

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

DNA diagnostic

The length of unknown fragments is compared to the length of standard 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)

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

Sugar-phosphate skeleton

DNA diagnostic

base pairs bounded by hydrogen bounds

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

DNA diagnostic

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

ladder PCR fragments

DNA diagnostic

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