(gene mutations, paternity, organs
compatibility for transplantations)
 Proteins
(gene expression)
DNA diagnostic
Basic steps:
› Cell lysis → DNA release
› Protein removal
 Protease
 Adsorption or extraction
› DNA precipitation by ethanol → impurities
› 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
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
› 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 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
The length of unknown fragments is compared to the length
of standard fragments
DNA diagnostic
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
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
base pairs bounded
by hydrogen bounds
DNA diagnostic
Temperature is a constant in each step
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
(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
DNA diagnostic
Exponential function
› Copies number of multiplying DNA region = 2n,
when n is number of cycles
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
DNA we want
to amplify
first cycle
(creating of two double stranded DNA
second cycle
(creating of four double stranded DNA
third cycle
(creating of eight double stranded DNA
DNA diagnostic
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
(ASO-PCR = PCR with allele specific oligonucleotides)
PCR with general primers – followed by PCR
product analysis
DNA diagnostic
Unknown mutation – complete analyses
searching for complete (exact) order of nucleotides in
amplificated DNA fragment
Known mutation – target analyses
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
 DNA analysis – Southern blot
RNA diagnostic
Real-Time PCR → PCR for qualitative and
quantitative analysis
(x DNA diagnostic – qualitative analysis only)
Reverse transcription
cDNA (complementary DNA)
Reverse transcriptase
› 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
› The more of target gene mRNA, the more of cDNA, the faster is
cDNA amplificated → gen is more expressed than other
(comparative analysis)