MUNI
MED
Genetics in Dentistry - Practice
Ing. Hana Holcova Polanská, Ph.D. Mgr. Lucie Válkové, Ph.D.
Practical Part
Practical Part
1. PCR
2. ELPHO
3. Evaluation of ELPHO
Practical implementation of PCR
volume in microtube: 25 |iL each sample
Composition (1 sample):
• 2 Primers 10 pmols (1.25 u±)
• MgCI2 25mM(4ui)
• dNTP mix (0.5 ul)
• Taq polymerase 1U (1 ul)
• Buffer DYNEX (2.5 uL)
• PCR H20 (12.5 u±)
• Template DNA 50ng (2u±)
1 drop of mineral oil
1. 95°C.....5 minutes
2. 95°C.....1 minute -
3. 60°C.....1 minute
4. 72°C.....1 minute _
5. 72°C.....7 minutes
6. 10°C.....10 minutes
Practical implementation of PCR
1) Prepare Master Mix for all samples you have by multiplying prescription by the number of samples (plus 1 extra sample).
2) Divide Master Mix to each microtubes by 23 [il.
3) Add one sample (2 piL) to each microtube.
Primer A     Primer B
MZSUL 1.25UL
Li.
Taq
dNTP mix polymerase
Buffer
0.5 UL
1 uL
PCR water
12.5 uL I <4-
MasterMix
Divide Master Mix to each microtubes by 23 uL.
Multiply prescription by the number of samples plus 1 extra sample {for 5 samples multiply by 6)
add 2 uiL sample: -j
Created in BioRender.com bio
Practical implementation of PCR
i *
1 drop of mineral oil
1. 95°C.....5 minutes
2. 95°C.....1 minute "1
3. 60°C.....1 minute
4. 72°C.....1 minute
5. 72°C.....7 minutes
6. 10°C.....10 minutes
35 x
o
How to Use a Micropipette
2
Plunger
Always hold the pipette vertically (tip pointing down).
Tip Ejector Button • Hold the pipette in a palm hanging from an index finger and Friction Ring operated by a thumb.
Body
Connecting Nut
• Choose optimal volume range!!! Never set the pipette outside of the range in either direction!!!
• Before pipetting, use a corresponding tip (according to the volume range of the pipette).
Tip Ejector
Tip Holder Tip Cone
• Always use a new sterile tip.
• While pipetting the same solution multiple times, use the same tip during the whole time.
Pipette Tip
• Tip ejector button on the side of the handle is for ejecting a used tip.
How to Use a Micropipette
Procedure:
• Set desired volume on the pipette. Horizontal line on display is a decimal point.
• Put a tip on a pipette (seal thoroughly) - not by hand!
• Pick up the pipette so that the finger rest of the handle is on the index finger and thumb can operate the two-position plunger.
• Aspirating: Press the plunger into the position „1" (pipette tip is in the air), immerse the tip into the liquid, slowly release the plunger.
• Dispensing: Immerse the pipette into the solution into which you want to dispense the aspirated liquid. Dispense by pressing the plunger into the position „1" finalize by pressing into the position „2" and take the tip out of the solution (still in position „2").
• Release the plunger, throw away the tip.
Plunger position:
Methods in Molecular Biology
Biological Materials
Biological material is everything that was or is a part or a product of a living organism
- dried herbal tea mixture
- apple core
- oak plank
- cat droppings/fur
- tube with SARS-CoV-2 virus, smallpox virus
- bodily fluids - urine, blood, plasma, serum, spit, ejaculate, phlegm
- tissues, cells
Methods in Molecular Biology
- Determination of nucleic acids
• PCR, RFLP-PCR - detection by ELPHO
• Real time PCR
• Sequencing
- Determination of proteins
• EUSA
• Western blot
• other methods based on antigen-antibody interactions
- Other molecular biology methods
Nucleic Acid Isolation
In native state from native material - in sufficient quantity and required purity.
NAs need to be devoid of all substances that would after lysis become a part of the crude substrate and that would impair the specific effects of enzymes used for further analyses.
Homegenizatton
^3
Lysis
Isolation of genomic DNA Isolation of RNA - focus on protection against degradation!
Homogenization/ Lysis
Separation
V
Phase Separation
Extraction/ Precipitation
Precipitation
Resuspension
I
Guanidinium-phenol
Water/TE
UNA p. CI
PCR - Polymerase Chain Reaction
Aim - acquisition of required specific sequence of genomic DNA without previous cloning Principle - multiple replications
• 25 to 35 cycles
• depends on temperature of reaction mixture
• amount of replicated DNA grows exponentially (2n)
PCR Protocol
Get the reagents      Prepare the mix     Set up conditions
Thermocycler
	
	^2
Analyze the gel       Negative result
13 SfeMii«^ *it«\ce
PCR
Multiple in vitro replication in a tube
Chain reaction based on DNA replication
Repeating cycles:
- denaturation (separation of dsDNA) - 96 °C
- annealing - primer binding - 50-65 °C
- elongation - synthesis of a new DNA strand - 72 °C
Denature DNA sample to separate DNA strands {94°C, 5 min)
Primers bind to DNA strands (30-65°C, 30 s]
Denature to separate DNA strands (94°C, 30 s)
Polymerase synthesizes new DNA strands (72'C, 45 s-2 min]
Region o(^~~ " interest 3'
I I I I I I I I I I I I I I I I I I I ' ' ' '........I I I 1 I I I I
98°C
II I I I I I I I I I II
I I I I I I I 1 I I I I I I I I I I I 5'
48 to 72°C
Denaturation
'temperature is increased to separate DNA strands
Primer
1 1 1 1 1 1 1 1 1 1......-v Annealins
3" -L        5'     Temperature is decreased
Primer
1 'I 1 1 1 1 1 1 I 1 1 1 1 1 1:.
I 68 to 72°C
to allow primers to base pair to complementary DNA template
TTTT" ¥^-rTTT-' I I I I I I I I I      II I I I I I I 5'"^
Extension
Polymerase extends primer to form nascent DNA strand
1st cycle —
111HIH! I! H11ITTT lllllllllllllllllll
22 = 4 copies
2nd cycle
rmrni	I1INI1I	
!!!!!!!!	1.......	!
........	anno	1
lull!!	tiiiiiii	I
V = 8 copies
3rd cycle —
initiiimimrm-..........I
.....1...........n
lllllllllllilllllll
lllllllllllllllllll lllllllllllllllllll
'.......1.......rn
IIIIIMllllllllllU
24 = 16 copies
4th cycle
.....11111111111
IIIIIIIIIIIIIIITT
limimHIHHI
lllllllllllllllll
"I"""......II
.....'........."
.....I.........'I
H...............
IIIIIIIMIIIIIIII
llllllllllilll
llllllllllllllllll
iiiiiiiiiiiniiin
->■ 30th cycle 231 = 2 billion copies
Exponential Amplification
Process is repeated, and the region of interest is amplified exponentially
IIIIIIIIIIM111,11
25 = 32 copies
PCR
DNA replication - in vitro (PCR)
• DNA polymerase
• thermostable (resists to temperatures up to 98 °C)
• Taq [Thermus oquoticus), Tth [Thermus thermophilus)
• primer
• short specific segments of DNA
• oligonucleotide 20-25 pb
• limiting the region for DNA amplification
• Mg2+ ions
• affect activity and precision of polymerase
• template DNA
dNTP
buffer (pH=8) temperature
DNA
TTTTTITT iririmn *
Primers
0s »„
} DNA Polymerase
DNA replication - in vivo
•  enzymes - helicase, primase, DNA polymerase, ligase...
DNA replication
DNA polymerase
<nxr
Lagging stand
Topoisomerase
JUKI
Parent DNA
Leading stand
I Download from Dreamstime.com
Q 41664959
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Video: https://www.youtube.com/watch?v=matsiHSuoOw and https://www.voutube.com/watch?v=oqeV72oYfD0
Gel Electrophoresis
separation method
principle - movement of charged molecules in direct current field (separation of molecules with different molecular weight)
speed of movement depends on the size of the total surface charge, size and shape of the molecule and its concentration in solution
DNA has uniformly negative charge—► in electric field moves from cathode to anode
parts of equipment
• electrophoretic container
• separation gel
• buffer
• direct current power supply
POWER SUPPLY
ELECTROPHORETIC BUFFER
CATHODE \
WELL
SAMPLEy'"^
HIGH MOLECULAR WEIGHT SPECIES
agarose (produced by seaweed - agar) EtBr-intercalates between bases, makes DNA visible under UV
ANODE
ANODE
-P^LOW MOLECULAR
WEIGHT ANALYTES
https://www.sciencedirect.com/topics/chemistrv/agarose-gel-electrophoresis Video: https://www.youtube.com/watch?v=vq759wKCCUQ
Gel Electrophoresis
• Size of DNA fragments can be determined with molecular-weight ladders (= restriction fragments of plasmid molecules or genome of bacteriophages, size of which was determined via sequencing)
GeneRuler™ 100 bp DNA Ladder
O'GeneRuler™ 100 bp DNA Ladder, ready-to-use
0.5 uj'lane, 20 cm length gel, 1XTAE.8V/cm,3h
RFLP - Restriction Fragment Length Polymorphism
Enzymatic cleavage of DNA in specific restriction site
Restriction endonucleases
Production of fragments with different lengths
Created fragments separated via gel electrophoresis
Use:
DNA mapping, analysis of DNA modifications, preparation of mutants
based on length and number of fragments we can observe differences in studied sequences, so called polymorphisms (polymorphisms are created by reconstruction of DNA strand, e.g. insertion, deletion, base substitution)
kinship analysis, determination of paternity, identification of persons
7kb    i 3kb
lOkb
RFLP
Restriction endonuclease
• sequence specific endonucleases (originated from bacteria)
• EcoRI {Escherichia coli), Hindll I {Haemophilus influenzae)
• blunt/sticky ends
• function:
• recognition of specific dsDNA sequence and subsequent restriction
(hydrolysis of phosphodiester bonds)
• recognition sequence
• 4-8 bp long
• character of palindrome =
same sequence of bases in both directions
5'
EcoRI
□ ■
i
3'
m G A A
□ EJ C
C & A A GIG
3' 5'
□ Q □ E3
5'
Psn
□ □
□
p c
♦
i
G C A G
□ 0Ü
□0GACG
C E3 □ ■ □
5'
Sma^
PCCCGGGÜ
□ □ □
□ □
□■□SGGGCCCPPIP
5'
m □ ■ m G p ■ □ □ c
A A
5' 3'
A A
C □
Gig
5'
5' Sticky ends
3
□ □ □ P
5
5'
G C
□
5' 3'
p e;
o c p G
3'
5'
G C A
[] E3u
3'
A C G
3'
3' Sticky ends
G & C p p ■ □
3' 5'
3'
□ □ ■ □ c c c
PigpGGG
5'
3
5' 3'
GGGOiU^U c c c p p ■ □
5
3'
5'
Blunt ends
qPCR - Quantitative Real-time PCR
Amplification Plot
• Polymerase chain reaction monitored in real time
• Quantification of DNA - amount of DNA is monitored during each cycle
• Detection of the DNA amount enabled by the presence of
fluorescent substrate
• Performed in a special cycler, which allows:
- Cyclical changes of temperature
- Fluorescence detection
- Monitoring of PCR progress in real time without the need to detect PCR products via electrophoresis
• qPCR is usually performed in 96-well plates, level of fluorescence is monitored in each well
• Highly sensitive and specific method
Amplification Plot
Amplification Plot
qPCR
Intensity of fluorescence is directly proportional to the amount of product created in the reaction
Product detection:
a) intercalating dyes - SYBR Green - non-specifically binds to dsDNA
b) sequence specific probe - short oligonucleotide with a dye and a quencher (TaqMan) - after its breakdown during DNA synthesis - rise of fluorescent signal (uses 5'-3' exonuclease activity of DNA polymerase)
SYBR
Denature •_
Polymerization
Siqnal detection (Polymerization completed)
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TaqMan
Annealing
Q ©
ine' \   Probe ~
Polymerization & strand displacement
Cleavage
Signal detection (Polymerization completed)
Video: https://youtu.be/YhXj5Yy4ksQ
DNA Sequencing
• determination of primary structure of DNA (sequence of nucleotides)
a) chemical method - degradation of nucleic acid chains via chemical agents (dimethyl sulfate, NaOH, hydrazine,..)
b) enzymatic method - specific inhibition of enzymatic synthesis of DNA
c) modern large format applications based on e.g. pyrosequencing (next generation sequencing)
• Product - strands of ssDNA, their relative sizes differing by one base (evaluation using ELPHO)
•   Input material - fragment of DNA with both defined ends
Maxam-Gilbert Sequencing
label
Sequence is derived from a DNA molecule which chemically degrades to fragments in places with a base of a specific type. These are subsequently separated using ELPHO.
Chemical agents - example:
- piperidine breaks glycosidic bond of A and G (A + G)
- hydrazine in the presence of NaCI reacts only with C
- NaOH at 90 °C strongly cleaves A and weakly cleaves C (A > C)
Requires radioactive labelling on one end of ssDNA.
Reaction is done in 4 tubes - in each only some types of bases are cleaved.
Mixture of differently long fragments ending in a place of a specific base is created —► evaluation using ELPHO, determination of a sequence of a given section.
I I I I I I I I I
OA
A>G
T+C
G>A    A>G T+C
1   I   I   1   I   I   1   I I
ACAATGCGT
Single stranded DNA template
Cleave at specific bases
Separate by gel electrophoresis antl detect labels
T
G
c
G
T A A C A
Video: https://www.youtube.com/watch?v= B5DJ8PL4E0
Sanger Sequencing
• Enzymatic method
• Based on principle of replication - end of DNA synthesis in the moment ddNTP is incorporated instead of dNTP
• ddNTP = analogue of dNTP, but lacks 3'-OH group on carbon
• ddNTP-terminator
• Reaction mixture (4x)
• DNA template
• primer
• Taq DNA polymerase - synthesis of DNA from 5' to 3' end
• buffer
• dNTP - abundant (to get fragments of all possible lengths)
• ddNTP - low concentration
• Evaluation - electrophoresis
/ G       A       T C
TGGTCGACGA
Í IZl3l4l3le|TI«<9ln
Video pro názornost: https://www.voutube.com/watch?v=wdS3iOTgbiM
Sanger Sequencing
• Modification -> fluorescently labeled ddNTP (4 different color labels) - reaction performed in one tube
• Capillary sequencing of DNA with fluorescently labeled ddNTP
® Reaction mixture
- Primer and DNA template    ► DNA polymerase
► ddNTPs with flourochromes ► dNTPs (dATP, dCTP, dGTP, and dTTP)
._ Hnmer
As i i i i u l l l 3'
-.................' -
Template ,—
ddNTPs
ddTTP-ddCTP-ddATP-ddGTP-
8 Primer elongation and chain termination
& I 1  I I
5't-
5'T-r
5- I  I  I I
5' i i i r
5' I I I I 5' I  I  I I
51 i i r
-r-r", 3'
■, 3
T-r1
* 3'
T, 3'
III* 3'
OH      OH      OH U^°-^J
HO-P-O—P-O
4,
IH       OK       OH "\^°^
<3> Capillary gel electrophoresis separation of DNA fragments
) Laser detection of flourochromes and computational sequence analysis
Chromalograph
NGS - Next Generation Sequencing
Sequencing of thousands to millions of sequences at the same time
Template DNA are fragmented sections few hundred bases long
Ends of fragments are enzymatically blunted and connected to oligonucleotides of specific sequence (= adapters)
Single fragments are separately amplified via PCR and in the next step sequenced in parallel
Fragmented input DNA
End Repair
dA Tailing
Adaptor Ligation
Size Selection
Amplification
						
Next Generation Sequencing						
						
						
Use:
- whole genome sequencing
- sequencing of chromosomes, plasmids, mt
- study of genetic variability mutational analysis
- transcriptome analysis
Video:
https://www.youtube.com/watch? v=CZeN-lgjYCo
https://www.youtube.com/watch? v=fCd6B5HRaZ8
Practical implementation of ELPHO
1. Prepare casting tray - combs and tape
2. Prepare gel (2%): weigh agarose and add it to Erlenmeyer flask, add TBE buffer (200 ml)
3. Weigh and boil in microwave.
4. After cooling to cca 40 °C add EtBr (1 pl/10 ml)
5. Cool down the gel (cca 30 min)
6. Remove combs and put into ELPHO container with TBE buffer (electrolyte)
7. Add size standard ladder (DNA + dye)
8. Prepare drops of loading dye on paraffin paper and mix with DNA samples.
9. Load samples into wells (max 15 pi)
10. Connect to power supply
11. Track the progress of DNA through the gel (40 min)
12. Visualisation under UV and evaluation
GeneRuler™ 100 bp DNA Ladder
O'GeneRuler'" 100 bp DNA Ladder, ready-to-use
bp IIU'0.5 |M     % bp
1/1000 45.0 9.0 16 900    45.0 9.0
■ 800
■ 700
m
45.0 9.0
45.0 9.0
45.0 9.0
500   115.0 23.0
400    40.0 8.0
40.0 8.0
I- 300 I- 200
. 1000 900 ■ 800
• 700
• 600
500
40.0 8.0
■ 100    40.0 8.0
0.5 Ma-'lane. 8 cm length gti. 1XT6E,5V/cm.1 h
5
0.5 pgflane, 20 cm length pel, 1XTAE,8Wcm,3h
Practical implementation of ELPHO
1. Prepare casting tray - combs and tape
2. Prepare gel (2%): weigh agarose and add it to Erlenmeyer flask, add TBE buffer (200 ml)
3. Weigh and boil in microwave.
4. After cooling to cca 40 °C add EtBr (1 u.l/10 ml)
5. Cool down the gel (cca 30 min)
6. Remove combs and put into ELPHO container with TBE buffer (electrolyte)
7. Add size standard ladder (DNA + dye)
8. Prepare drops of loading dye on paraffin paper and mix with DNA samples.
9. Load samples into wells (max 15 u.1)
10. Connect to power supply
11. Track the progress of DNA through the gel (60 min)
12. Visualisation under UV and evaluation
GeneRuler™ 100 bp DNA Ladder
O'GeneRuler'" 100 bp DNA Ladder, ready-to-use
bp IIU'0.5 |M     % bp
1/1000 45.0 9.0 16 900    45.0 9.0
■ 800
■ 700
m
45.0 9.0
45.0 9.0
45.0 9.0
500   115.0 23.0
400    40.0 8.0
40.0 8.0
I- 300 I- 200
. 1000 900 ■ 800
• 700
• 600
500
40.0 8.0
■ 100    40.0 8.0
0.5 Ma-'lane. 8 cm length gti. 1XT6E,5V/cm.1 h
5
0.5 pgflane, 20 cm length pel, 1XTAE,8Wcm,3h
Recommended Literature for Self-study
Practical implementation of ELPHO
Electrophoresis of restrictive fragments after enzymatic cleavage
Heterozygote Homozygote (cleaved)
Thank you for you attention
ulij JĽm