Some mistakes have been corrected.

Notes to students: please, take attention on restrictases

and PCR part.

Next presentation „Methods of DNA study II“ (english

version) will be released on the 22nd of April.
 21st April 20200

METHODS OF DNA ANALYSIS I

A lecture to molecular biology practices (summer semester)

Mgr. Petr Daniel

Department of Biochemistry, Cell and Molecular biology 3. LF UK

petr.daniel@lf3.cuni.cz
 Practices of MB, summer semester 2020

 The molecular biology practices are held in distinct form of

study due to coronavirus pandemic this year 

 Please, see attached video files in Vyuka application

To get credits, it is necessary…

➢ ..make and send me a protocol (can be done in a group of
maximally of 3 people)
➢ the file with protocol will be added to Vyuka application
➢ information will be discussed in practice presentation
 Diagnostics

 DNA – gene sequence, repetitive sequence,

non-coding sequence

 RNA gene expression

 Proteins

Sources of nucleic acids

 DNA of particular gene

➢ All nuclear cells
 RNA of particular gene
➢ Only those cells, that express the gene of interest
 Why we use nucleic acid diagnostics

 High sensitivity
 Accuracy
 Fast
 Low price (instruments, chemicals)
 Quantity
 Automatization
 DNA polymorphism and mutations

 Allele polymorphism
➢ Physiological function, frequence > 1%
➢ Predisposition to polygenic diseases

 Mutation
➢ Patological function, frrequence < 1% (selection)
➢ The cause of monogenic diseases
➢ Occurs spontaneously in the germ cells of one of the
parent (např. monogenic autism – mutation in one of
many genes)
 Types of DNA polymorphism

 The variability in DNA sequence among individuals of the same

species (99,9% for people x 98,5% between human and
chimpanze)

 SNPs (snips) – single nucleotide polymorphisms

➢ The change in a single nucleotide only
➢ Usually 2 alleles in a population
➢ Evenly distributed in human genome – convenient for gene
maping
➢ Detection - RFLP, sequencing, DNA chips

5´ AGCTCAGAAATC 3´
3´ 5´ Allele 1

5´ AGCTCACAAATC 3´
3´ 5´ Allele 2
 Minisatelites (VNTRs) – variable number of tandem repeats
➢ The unit is long (up to 25 bp)
➢ Mostly in telomeres, not evenly distributed in genome
➢ Total length up to several kb

 Microsatelites (STRs) – short tandem repeats

➢ The unit is short (2-6 bp) and repeated10-30 times
➢ 3 millions STR in a human DNA
➢ Shorter units predominate (TATATATA, CTGCTGCTGCTG)
➢ Total length up to 300 bp – optimal for detection by PCR

5´ TCTGAGAGAGGC 3´
3´ 5´ Allele 1

5´ TCTGAGAGAGAGAGAGGC 3´
3´ 5´ Allele 2, Allele 3…..
 Charakteristics of DNA diagnostic

Detection of polymorphism of particular gene

 TARGETED ANALYSES
➢ Localization and whole sequence of gene is known
➢ Mutation of gene is known
➢ Checkup of family members is not necessary

 COMPLETE ANALYSES
➢ Localization and whole sequence of gene is known
➢ Mutations are not described
➢ Checkup of family members is necessary
 What is detected?

 Monogenic and polygenic inherited diseases

 Cancer typization
 Detection of viruses and bacteria
 Progression of disease
 Identification of persons in forensic medicine
 HLA-typization for organ transplantation
…
 Prevention - : - preimplantation
- prenatal
- presymptomatic
 Progression of cancer and personalized therapy

Non-small cell lung cancer (NSCLC)

➢ Mutations in the gene for epitelial growth factor receptor (EGFR)
(exon 19 deletion, and L858R)

➢ Tyrosine kinase inhibitors (TKI)

✓ 1st generation: Gefitinib, Erlotinib
✓ 2nd generation : Afatinib

9-15 months T790M

✓ 3rd generation: Osimertinib

several months KRAS,

EGFR
 Detection of mutations and polymorphism
RNA
isolation
DNA isolation

Reverse
transcription

DNA DNA
fragmentation amplification

Separation Fragmentation Separation Sequencing

Southern blot Separation

RFLP
 DNA isolation
 Choosing the method – quality and quantity of DNA
➢ Kit (binding DNA on silicate column and washing it from
impurities)
➢ Home-made metods (i.e., phenol-chloroform extraction,
other protocols)

 Basic steps :
➢ 1. Cell lysis (releasing the cell components into solution)
➢ 2. Removing of macromolecules (proteins/RNA)
 Incubation with RNase A (RNA degradation)
Proteinase K (protein degradation)
➢ 3. DNA precipitation with ethanol (removing salts and small
organic compounds)
➢ 4. DNA solving in water or buffer
 DNA isolation

 Cell lysis
➢ Mechanically (grinding, vortexing)
➢ Physically (ultrasound, freeze/thaw cycles)
➢ Chemically (detergent) – in commercial kits

(these methods are generally combined)

 Chemical lysis – example of the lysis buffer:

› Buffer, TRIS-HCl, pH 8,0 – keeping optimal pH
› Chelatator, 1 mM EDTA – inhibits enzymes
› Osmolarity, 0.2 mM CaCl2
› Detergent, i.e. 0,001% SDS (a strong ionic detergent)
0,001% Triton X-100 (nonionic detergent)
Restriction fragment length polymorphism, RFLP
 Based on the enzymatic activity of class II restriction enzymes
(abbr. restrictase)
➢ Restriction = the part of restriction-modification system of
bacteria, to protect bacteria against foreign viral DNA
➢ Nuclease = an enzyme that degrades a nucleic acid
 Exonuclease = cleaves nucleic acid from one of the end
❖ 5' → 3' exonuclease
❖ 3' → 5´exonuclease

 Endonuclease = cleaves inside

a nucleic acid
❖ Specifically

❖ Nonspecifically

 Some restriction sites are polymorphic

 105 RFLP / genome

 Features of class II restrictases
 An enzyme that recognizes specific cleavage sequence
(„restriction site“) in double-stranded DNA and then it performes
a cleavage of phosphodiester bond within or in close proximity
of that site (acts like a molecular scissors)

 Under the optimal conditions an

enzyme cleaves all restriction sites
 Available more than 700 enzymes
 Name
latin name phyllum order of gene

EcoRI Escherichia coli RY13 I

BamHI Bacillus amyloliquefaciens H I

XhoI Xanthomonas holcicola I
https://pdb101.rcsb.org/motm/8
 Features of class II restrictases
 Sequence specific nuclease activity, no ATP is need
➢ Restriction site is basically a short palindromic sequence (4-8 nt)

m
5´ …. G A A T T C …. 3´ 5´ …. G A A T T C …. 3´
EcoRI
3´ …. C T T A A G …. 5´ 3´ …. C T T A A G …. 5´
m

Human DNA Escherichia coli DNA

 Methyltransferase activity is completely absent

 Homodimer
 Enzymatic activity = unit (1 µg DNA in 1 hour, optimal conditions)
 Star activity – nonspecific activity, not optimal conditions
 After DNA cleavage, we get fragments with different ends:
➢ Sticky ends (cohesive) (Fig. A)
➢ Blunt ends (Fig. B)

Blunt end
P
A
P

5‘ Sticky end
P
P

P
B
P

P
The average length of DNA fragment after the cleavage

N = 4n

N – average length of DNA fragment of random sequence

n – number of nucleotides creating the restriction site

Example. A restrictase recognizes 4 nucleotide site.

N= 44 = 256 3.2Gbp/256bp = 12,500,000 fragments

Example. A restrictase recognizes 6 nucleotide site.

N= 46 = 4096 3.2Gbp/4096bp = 781,250 fragments
 Southern blot
 Transfer of DNA fragments from agarose gel into
nylon membrane
 sir Edwin Southern
 Hybridization probe can not reach DNA in the
gel https://en.wikipedia.org/wik
i/Edwin_Southern

 Gel electrophoresis of human

chromosomal DNA after
cleavage with XbaI enzyme
(figure on the left)
 Smear of DNA fragments (we
can not distinguish between
individual fragments)
Southern blot
 Example of RFLP – sickle cell anemia
 Autosomal reccesive
 Incomplete dominance HbS
 Mutated allele HbS is differing in
one nucleotide (A is changed for
T)
 Codon GAG(Val) is changed to
GTG (Glu)

Zápis: c.20A>T, p.Glu7Val  Protein hemoglobine S makes

fibers that change the shape of
erythrocytes
 Bsu36I (MstII) recognizes
 Cause vein damage (pain)
“CCTNAGG“ sequence
 Anemia, high probabilty of
infections
 Resistance to malaria
PCR = polymerase chain reaction

 Karry B. Mullis – 1983 conceived PCR

method
 A nobelist, loved surfing and LSD
 He worked in Cetus corporation
 He got 10,000$ from Cetus corp, and
Cetus corp sold the patent for
250,000,000$ to other company
 In vitro synthesis of DNA segment
 Cycles of Denaturation → Reasociation
→ Synthesis
 Exponential yield of product
 Basic components of PCR

 DNA template
 Pair of primers (forward primer and reverse primer)
 Free nucleotides(dNTPs)
 Thermostable DNA dependent DNA polymerase
 Buffer
 Activator (Mg2+)

Other components – enhanced the yield

➢ Betaine, DMSO – GC hairpins
➢ Formamide – eliminates non-specifc binding of primers
➢ Spermidine – protects DNA against radicals
➢ BSA – bind to the wall of PCR tube
➢ Tween 20 – eliminates the effect of residual SDS detergent
in isolation buffer
 Template DNA
 The amount of chromosomal DNA for typical PCR reaction is
100 – 500 ng
 Avoid of contaminations, especially NUCLEASES
➢ Elsewhere in our environmentr (on the hands)
➢ Hard to inactivate
➢ Use only clean tips (tested on presence of nucleases)
➢ Often change glows
➢ Must be mixed in PCR box

RNA – binds to Mg2+

DNA – samples must be prepared separately (cross-
contamination)
 Determination of DNA purity and concentration

 Spectrophotometry
➢ DNA/RNA bases absorb UV (236-300 nm)
➢ Absorbance maximum:
for nucleic acids (DNA, RNA) 260 nm
for proteins 280 nm
for small organics molecules 230 nm

→ DNA concentration: at 260 nm

→ DNA purity is done by ratio 260/280 nm
(1,6-2,0) and 260/230 nm (2,0 – 2,5)
 Isolated DNA – home method
nečistoty

DNA/RNA peak

proteins

200 nm 300 nm 400 nm

 After DNA purification

Determination of DNA quality and concentration

 Gel electrophoresis with fluorescent dye

(not so precise)

➢ DNA shines after binding of fluorescent dye

(green)
➢ We compare the signal intensity of DNA of
known concentration with DNA of unknown
concentration
 Agarose gel electrophoresis
A separation method

 DNA harbors negatively charged

phosphate groups → will migrate
from cathode (-) to anode (+)

 The speed movement is

dependent on the fragment
length indirectly

Separation of DNA fragments (RNA, proteins) according to their

length on the basis of movement of charged molecules in the
electric field
 Gel – 3D polymeric net  TAE / TBE + agarose

 Agarose / polyacrylamide
➢ Different resolving ability:
polyacrylamide is able to distinguished DNA
fragments differing in one nucleotide
agarose separates fragments differing in
minimally 10 nucleotides

 Ethidiumbromide – a dye added to a gel

› Binds into DNA structure
› After UV exposition it emits photons (shines)
 -

A way of movement
+

MW ladder DNA
(bp = base pair)
fragments