kamila balušíková. dna – sequence of genes, repetitive sequence of noncoding regions rna ...
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Methods of DNA and RNA study
Kamila Balušíková
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Diagnostics
DNA – sequence of genes, repetitive sequence of noncoding regions
RNA Proteins
gene expression
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Source of nucleic acids
DNA of certain gene – all nuclear cells
RNA of certain gene – only the cells, where this gene is
expressed
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DNA diagnostic
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What can we detect ?
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
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DNA polymorphisms
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
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Characteristics of DNA diagnostics
Detection of certain polymorphism of the predisposition gene
TARGET ANALYSES COMPLETE ANALYSES
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Target analyses
The localization and the whole sequence of a gene is known
The mutation of the gene is known
An examination of family membersis not needed
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Complete analyses
The localization and the whole sequence of a gene is known
Mutations of the gene are unknown
An examination of family members is necessary
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Principle of testing
DNA isolation
PCR (amplification of a DNA region)+ other analyses
result visualization
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DNA izolation
Basic steps:
› Cell lysis → DNA release› Protein removal
ProteaseAdsorption or extraction
› DNA precipitation by ethanol → impurities
removal› DNA dissolution in water or buffer
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DNA purity and concentration
Spectrofotometryabsorption maximum
for nucleic acids 260 nmfor proteins 280 nm
→ DNA concentration: at 260 nm→ DNA purity is calculated by ratio 260/280 nm
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DNA purity and concentration
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) – comparison of two light intensities
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Gel electrophoresis- separation method
Separating of DNA fragments (RNA, protein molecules) according to their molecular weight (size) Principle: movement of charged
molecules in electric field
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Gel electrophoresis
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)
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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)
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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)
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MW marker(bp = base pairs)
DNA fragments
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PCR (polymerase chain reaction)
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
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PCR (polymerase chain reaction)
Basic compounds in PCR reaction
DNA sample Pair of primers Free nucleotides (dATP, dTTP, dCTP, dGTP) DNA polymerase with buffer
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Primers
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
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Sugar-phosphate skeleton
base pairs bounded by hydrogen bounds
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Steps of PCR
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)
Temperature is a constant in each step
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Amplification
Exponential function› Copies number of multiplying DNA region = 2n,
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
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
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PCR types
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
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PCR subtypes
Nested PCR (includes two successive PCR reaction)
Multiplex PCR (employs two or more PCR in same time – one reaction mix)
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PCR product analysis(PCR with general primers)
Unknown mutation – complete analyses
Sequencingsearching for complete (exact) order of nucleotides in amplified DNA fragment
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PCR product analysis(PCR with general primers)
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)
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Gene expression levels – mRNA– proteins
mRNA – Real-Time PCR, Northern blot Proteins – Western blot
Detection of certain gene expression
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Gene expression on mRNA level
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
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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)