Polymerase chain reactions,

Real time polymerase chain reactions;

Case discussions (Sequencing of HCV,
HBV and HIV, interpretation and reporting)
Next generation sequencing and disease
management

Marine Karchava, MD, PhD.

Petre Shotadze Tbilisi Medical Academy
Lecture # 3
Date: 08.04.2022
Location: Infectious Diseases, AIDS and Clinical Immunology
Research Center
 Question # 1
Chromosome is the thread-like structure with
the DNA molecule in it

• True

• False
 Role of DNA

Deoxyribonucleic acid (DNA) is a nucleic acid that

contains the genetic instructions for the
development and function of living things

• Replication
• Encoding information
• Mutation/recombination
• Gene expression
 Nucleic acids in eukaryotic cells
• Nucleic acids aka DNA or RNA are critical molecules of
life.
• Deoxyribonucleic acid (DNA) resides in the nucleus of
eukaryotic cells and maintains all the information
necessary for maintenance of the organism and for
transfer of the information to successive generations.
• Ribonucleic acid (RNA) carries information from DNA
to the cytoplasm of a cell and directs synthesis of the
proteins necessary for the function of the organism.
 Structure of DNA and RNA

• DNA is a long, double-

stranded polymeric
molecule (dsDNA) that
exists predominantly in the
form of a right-handed
double helix.
• Each single stranded DNA
molecule (ssDNA) is
composed of a small
number of building blocks.
• The backbone of the
dsDNA polymer is the sugar
deoxyribose connected by
phosphate groups
DNA structure
 Repeating backbone of DNA;
complementary base pairs
• A single-stranded
DNA chain.

• Repeating
nucleotide units
are linked by
phosphodiester
bonds that join the
5′ carbon of one
sugar to the 3′
carbon of the next
 Repeating backbone of DNA;
complementary base pairs

• Purine and pyrimidine

bases and the
formation of
complementary base
pairs.
• Shaded bars indicate
the formation of
hydrogen bonds.
 Structure of DNA
• Phosphodiester bonds between the 3′ carbon of one sugar
ring and the 5′ carbon of the next give the backbone its
invariant structure and its 5′ to 3′ directionality.
• Linked to the 1′ carbon of each sugar is one of four possible
bases: thymine (T) and cytosine (C) (pyrimidines), and
adenine (A) and guanine (G) (purines).
• The building blocks of the single-stranded polymer are the
four deoxyribonucleotide triphosphates (dTTP, dCTP, dATP,
dGTP), each consisting of a sugar molecule, a triphosphate
group, and one base.
 DNA characteristics
• DNA is an extraordinarily stable molecule,
losing its normal conformational structure
only at extremes of
Heat
PH
Destabilizing agents
• Both sugar and phosphate groups are
hydrophilic, forming stable hydrogen bonds
with surrounding water molecules in solution.
 Structure of RNA
• RNA differs from DNA in chemical
composition, structure, and
function
• RNA exists predominately as a
single-stranded molecule
• In RNA the sugar is ribose,
containing a hydroxyl group at the
2′ position, and thymine is
replaced by the methylated uracil
(U).
• The structure of RNA is more
irregular, owing to the single-
stranded nature of the molecule,
but it may contain some
DNA vs RNA
 DNA basics
• Gene
Genes are small sections of
DNA within the genome that
code for proteins
• Genome
Genome is the genetic material
of an organism, that consisted
of thousands of genes
• Locus
locus is a specific, fixed
position of particular gene on a
chromosome
 DNA basics
• Protein • Coding region (Exon)
The coding region of a gene, also known as the CDS
protein is composed of (from coding sequence), is that portion of a gene's
amino acids, which are DNA or RNA that codes for protein.
organic compounds made • Non coding region-(Intron)
of carbon, hydrogen, Noncoding DNA sequences are components of an
organism's DNA that do not encode protein
nitrogen, oxygen or sulfur. sequences
 DNA steps
• DNA replication
• DNA transcription
• DNA translation
 DNA replication
• During the DNA duplication
process, uses each strand of
the parent molecule to
direct the synthesis of a
daughter strand
• Base sequence of the parent
strand dictates the
sequence of the daughter
strand
• Resulted new strand is
exactly the same base pair
sequence as parent strand
 DNA transcription
• DNA produces complimentary form of RNA
• If RNA carries the code from the DNA in the cell nucleus to
the cytoplasm where amino acid synthesis takes place, this
type of RNA is called messenger RNA (mRNA)
• Messenger RNA is synthesized from only one strand (coding
strand) of the DNA gene
Replication-transcription-translation
 History of PCR
• Definition of PCR-
Polymerase chain reaction is a exponentially
progressing synthesis of defined target
DNA/RNA sequences in vitro

• Developed by Kary Mullis in 1983

• Kary Mullis and Michael Smith were awarded
Nobel Price in chemistry in 1993
 Steps of PCR
• Step # 1- Nucleic acid extraction
• Step # 2- Nucleic acid Amplification
• Step # 3- Nucleic acid detection
 PCR Step # 1
Nucleic Acid extraction
• DNA and RNA extraction from blood/plasma/
serum etc
 PCR Step # 1
Nucleic Acid extraction
• Column based extraction
• Magnetic bead extraction
 PCR Step # 1
Nucleic Acid extraction
Extraction process frees DNA/RNA from the white
blood cell and then separates it from cellular fluid
and proteins so you are left with pure DNA/RNA.

The three basic steps of DNA extraction are

• 1) lysis (temperature incubation)
• 2) precipitation (centrifugation)
• 3) purification (centrifugation)
 PCR Step # 1
Nucleic Acid extraction
• Manual extraction methods
 PCR Step # 1
Nucleic Acid extraction
• Automated extraction
 Product of PCR step 1
• Purified DNA
• Purified RNA
• Purified total Nucleic acid
 PCR Step # 2
Amplification
• Polymerase chain reaction (PCR) is an efficient and cost-
effective molecular tool to copy or amplify small segments of
DNA or RNA

• PCR combines the principles of

 complementary nucleic acid hybridization
 nucleic acid replication
repeatedly through numerous cycles

• It results in the production of the specific target DNA/RNA

sequences by a factor of 10^7 within a relatively short period.
PCR Step # 2
Amplification
 PCR Step # 2
Amplification
Components of amplification mixture
• DNA /RNA ( from step # 1)
• dNTPs
• PCR buffer
• Primers (for and rev)
• Taq polymerase
• H2O
 PCR Step # 2
Amplification
The three stages of a PCR test are

• Denaturation- when the double-stranded template

DNA is heated to separate it into two single strands
• Annealing -when the temperature is lowered to
enable the DNA primers to attach to the template
DNA
• Extending/elongation – when the temperature is
raised and the new strand of DNA is made by the Taq
polymerase enzyme.
PCR Step # 2
Amplification
 PCR Step # 2
Exponential Amplification

PCR Cycles
 PCR Step # 2
Amplification
• Template RNA- RT-PCR
• RT-PCR is a technique that includes combining
reverse transcription of RNA into DNA (in this
context called complementary DNA or cDNA).
• Amplification step has additional step on low
temperature 37C.
PCR Step # 2
Amplification
 PCR Step # 2
Amplification
• PCR thermal cyclers
 PCR Step # 3
Detection
• Product is amplified DNA that need to be
visualized.
• A common method of visualization is via
agarose gel electrophoresis.
• This method involves separating amplification
product fragments via electrophoresis (electric
charge) and staining with an dye such as
ethidium bromide or subsequent detection
using a UV light source and imaging system.
 PCR Step # 3
Detection
• Agarose powder
• Size marker (DNA ladder)
• Ethidium bromide
• TE buffer
• Gel electrophoresis system
• Gel photo documentation
 PCR Step # 3
Detection
• Real-time PCR uses specialized thermocyclers
that detect the fluorescent signal in each well.
• This signal is indicative of the amount of
double-stranded DNA within the reaction tube
or well.
• The signal, in relative fluorescent units, is
plotted by the thermocycler software versus
cycle number
PCR Step # 3
Detection
 PCR Step # 3
Detection
• Equipment
 PCR Step # 3
Detection
• Reverse hybridization methods
COBAS Ampliprep/Cobas Taqman HCV
quantitative test
COBAS Ampliprep/Cobas Taqman HCV
qualitative test
COBAS Ampliprep/Cobas Taqman HCV
qualitative test
COBAS Ampliprep/Cobas Taqman HCV
qualitative test
COBAS Ampliprep/Cobas Taqman HCV
qualitative test
 PCR Step # 3
Detection

• Quantitation- viral load

• Specific- presence/absence assay
• Multiplex PCR- More than one target can be
amplified by using multiple primer pairs in a
reaction mixture
 Reporting
• Hepatitis C RNA
• Hepatitis B DNA
• Hepatitis Delta RNA
• HIV 1, 2-RNA
• Herpes simplex viruses 1 and 2; varicella zoster
virus; mumps virus; enterovirus; human
parechovirus
• Leishmanis donovani
• Abacavir
 Nucleic acid extraction
• https://www.youtube.com/watch?v=7QQOivJ
gHkM
 One step vs two step PCR
• https://www.youtube.com/watch?v=JGoGhp6
vcSE&t=7s

• https://www.youtube.com/watch?v=iQsu3Kz9
NYo
DNA Mutation
 Reading
• Clinical Diagnosis and Management of
Laboratory Methods by Richard A.
McPherson, MD, MSc and Matthew R. Pincus,
MD, PhD
• Pages 1316-1326
• Electronic book is uploaded in TMA.INI.GE
Topic #2
DNA Sequencing and disease
management
 Personalized medicine

• What is personalized therapy?

• What is personalized medicine used for?
• Why is personalized medicine important?
• What is an example of personalized medicine?
 Personalized medicine is a move away from a
'one size fits all' approach to the treatment and
care of patients with a particular condition
Sanger sequencing
 DNA sequencing history
• Maxam ,Gilbert and Sanger, simultaneously designed a in vitro procedure
similar to the natural process of DNA replication
• Both teams shared 1980 Nobel Prize
• Frederick Sanger’s method became the standard because of its practicality
 Essentials of DNA sequencing
• DNA sequencing is the technology, which
provides us with the detailed structure of the
the nucleotides.
• Using this technology, we can locate specific
mutations sequences
• Make comparisons between homologous
genes across species
 Human Genome Project
• The Human Genome
Project (HGP) was an
international scientific
research project
• Initiated in 1990
• Identifying and
mapping all of the
genes of the human
genome
Human Genome Project
 Steps of DNA sequencing
• Step # 1- Nucleic acid extraction
• Step # 2- Nucleic acid Amplification
• Step # 3- Nucleic acid detection (agaroze gel)
• Step # 4- PCR product purification (alcohol…
cephadex, sodium…)
• Step # 5- Sequencing reaction
 Steps of DNA sequencing
• Denaturation of DNA product using heat
• Primer bonding to the DNA template strands.

Primer is specifically constructed so that its 3' end

is located next to the DNA sequence of interest.

Either this primer or one of the nucleotides

should be radioactively or fluorescently labeled so
that the final product can be detected.
PCR primers vs Sequencing primers
 Sanger reaction mixtures in
4 tube method

• Nucleotides
• Primer
 Strand of nucleic acid
 Serves as starting point for DNA replication
• Polymerase
• Buffer
Nucleotides
Nucleotides
 Nucleotides
ddNTP- dideoxynucleotide dNTP- dieoxynucleotide
• ddGTP- • dGTP-
• ddTTP- • dTTP-
• ddATP- • dATP-
• ddCTP- • dCTP-

Two different types on nucleotides are added in the reaction all four tubes along with primers and
polymerase
 Nucleotides

• During the DNA synthesis step normal nucleotide (dNTPs) are added on to the
growing chain by the DNA polymerase.
• On occasion a ddNTPs are incorporated into the chain in place of a dNTPs,
which results in a chain-terminating event.
• In this case, if we looked at only the "G" tube, we might find a mixture of the
following products
 Sanger sequencing
• The key to this method, is that all the
reactions start from the same nucleotide and
end with a specific base G or T or A or C
• Thus, bands of all different lengths are
produced
• After completion of reaction, the DNA is once
again denatured for electrophoresis
DNA sequencing in G tube
DNA fragment
DNA sequencing
Sanger sequencing equipment

• SeqStudio™ Genetic
Analyzer System

• 3500xL Genetic Analyzer

Chromatogram
Hepatitis C NS5A region sequence
> Sample_28614

CACTATGTGCCTGAGAGCGACGCTGCGGCACGCGTTACTCAGATCCTCTC
CAGCCTTACCATCACTCAGCTGTTGAAGAGGCTCCATCAGTGGATCAATG
AGGACTGCTCCACGCCGTGCTCCGGCTCGTGGCTAAGGGATGTTTGGGA
TTGGATATGCACGGTGTTGACTGACTTCAAGACCTGGCTCCAGTCCAAGC
TCCTGCCGCGAATGCCGGGAGTCCCTTTTTTCTCGTGTCAACGCGGGTAT
AAGGGAGTCTGGCGCGGAGACGGCATCATGCACACCATCTGCCCATGTG
GAGCACAGATTACCGGACATGTCAAAAACGGTTCCATGAGGATCGTTGG
GCCTAAGGCCTGCAGCAACACGTGGCACGGGACGTTTCCCATCAACGCA
CACACCACGGGTCTCTGCACACCCTCCCCGGCGCCGAACTATTCCAGGGC
GCTGTGGCGGGTGGCCGCTGAGGAGTACGTGGAAGTTACGCGGGTGGG
GGA
HCV geno2pheno
Sample of Hepatitis C NS5A region
sequence analyses report
 HIV 1 pol gene sequence
Patient 1
> HIV pol gene

AAATTTTCCTCAGAGCAGACCAGARCCATCAGCTCCACCAGCAGAGAGCAAATCACTCTTTGGCAACGACCC
CTTGTCRCARTAAGAGTRGGRGGACAGTTAAGRGARGCTCTATTAGATACAGGAGCAGATGATACAGTATTA
GAAGACATAGATTTGCCAGGAAAATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAG
ACARTATGATCAGATACTTATAGAAATCTGTGGRAAAAAGGCTATTGGTACAGTATTAGTAGGACCTACCCCTG
TCAACATAATTGGRAGAAATATGTTGACTCAGCTTGGTTGTACTTTAAATTTTCCAATAAGTCCTATTGAAACT
GTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAGGTYAAACAATGGCCATTAACAGAAGAAAAAAT
AAAAGCATTAACAGCAATTTGTGATGARATGGAAAAGGAAGGAAAAATTTCAAAAATAGGGCCTGAAAATC
CATACAATACTCCAATATTTGCTATAAAGAAAAAGGACAGCACTAAGTGGAGRAAATTAGTAGATTTCAGGGA
GCTCAATAAAAGAACTCAGGATTTYTGGGAAGTTCAATTAGGAATACCCCATCCAGCGGGTTTWAARAAGA
AAAAATCAGTAACAGTACTAGATGTGGGGGATGCRTATTTTTCARTCCCCTTAGATGAAAGYTTCAGAAAGTA
TACTGCATTTACTATACCAAGTACAAACAATGARACACCAGGGATCAGATATCAGTACAATGTACTTCCACAGG
GATGGAAGGGATCACCAGCAATATTTCAGAGTAGCATGACAAAAATCTTAGAGCCCTTTAGATYAAAAAATC
CAGAAATAGTTATCTATCAATACATGGATGACTTGTATGTAGGCTCTGATTTAGAAATAGAACARCATAGAACA
AAARTAGAGGAGTTRAGAGCTCATCTATTGAGCTGGGGRTTTACTACACCAGATAAAAAGCAYCAGAAAGA
ACCTCCATTTCTTTGGATGGGGTATGAACTYCATCCTGACAAATGGACAGTCCAGCCTATAGTACTGCCAGAT
AAAGACAGCTGGACTGTCAATGATATACAGAAATTAGTGGGAAAACTAAATTGGGCAAGTCAGATCTATCCA
GGGATYAAAGTAAARCAATTGTGTAAACTCCTCAGGGGAACCAAAGCACTGACAGAWATAGTGACACTGAC
TCAGGAAGCAGAATTAGAATTGGCAGAGAACAGAGAGATTCTAAAAGAACCTGTGCATGGAGYATAYTATG
Report Patient 1
Report Patient 1
 HIV 1 pol gene sequence
Patient 2
> HIV
AGGAAATTTTCCTCAGAGCAGACCAGAGCCATCAGCCCCGCCAGCAGACCTCAAATCACTCTTTGGCA
ACGACCCCTTGTCACAGTAAGAATAGGAGGACAGCTAAGAGAAGCTCTATTAGATACAGGAGCAGATG
ATACAGTATTAGAAGAAATAAATTTGCCAGGAAAATGGAAACCAAAGATGATAGGGGGAATTGGAGG
TTTTATCAAAGTAAGACAGTATGATCAGATACTTATAGAAATTTGTGGAAAAAAGGCTATAGGTCAGTAT
TAGTAGGACCTACCCCTGTCAACATAATTGGAAGAAATATGTTGACTCAGCTTGGCTGTACTTTAAATTT
TCCAATAAGTCCTATTGAAACTGTACCAGTAAAATTAAAACCAGGAATGGATGGCCCAAAGGTTAAACA
ATGGCCATTAACAGAAGAGAAAATAAAAGCATTAACAGAAATTTGTGATGAGATGGAGAAAGAAGGA
AAAATTTCAAAAATTGGGCCTGAAAACCCATACAATACTCCAATATTTGCTATAAAGAGAAAGGACAGC
ACAAAGTGGAGAAAATTAGTAGATTTCAGGGAACTCAATAAAAGAACTCAGGACTTTTGGGAAGTTC
AATTAGGAATACCCCATCCAGCGGGTTTAGAAAAGAAAAAATCAGTAACAGTACTAGATGTGGGAGAT
GCATATTTTTCAGTTCCTTTAGATGAAAGCTTCAGGAAGTATACTGCATTCACTATACCAAGCACAAACA
ATGAGACACCAGGAACCAGATATCAGTAYAATGTACTTCCACAGGGATGGAAAGGATCACCAGCAATAT
TCCAGAGTAGCATGACAAAAATATTAGAGCCCTTTAGATCACAAAATCCAGACATAGTTATCTRTCARTA
CATGGATGAYTTGTATGTGAGCTCTGACTTAGAAATAGGGCAACATAGAATAAAAATAGAGGAGTTGAG
ARCTCATCTATTGAAATGGGGATTTATTACACCAGACAAAAAGCATCAGAAGGAACCTCCATTTCTTTG
GATGGGATATGAACTCCATCCTGACAAATGGACAGTCCAGCCTATAAAGCTGCCAGATAAAGACAGCT
GGACTGTCAATGATATACAGAAATTAGTGGGAAAACTAAATTGGGCAAGTCAGATTTATCCAGGGATTA
AAGTAAGACAATTGTGTAAACTCCTCAGGGGAGCCAAAGCACTGACAGATATAGTGACACTGACTGAG
GAAGCAGAATTAGAATTGGCAGAGAACAGAGAGATTCTAAAAGAACCTGTGCATGGAGTATATTATGA
Report
Report
NEXT generation sequencing
 NGS- Next generation sequencing
technology
• NGS technology has revolutionized genomic
research through
– increased throughput and decreased costs
– It is now becoming possible to sequence an entire
genome at a turnaround time acceptable for clinical
applications.
– Currently the diagnostic clinical utility of a complete
human genome sequence is still being evaluated, but
the knowledge of genetic and genomic sequence
alterations has great potential to impact clinical
pathology.
 NGS- Next generation sequencing
applications
• Direct sequencing is being used to determine
viral types (e.g., human papillomavirus),
bacteria, and other pathogens.
• The greatest advances are made in cancer
oncology to identify key mutations in oncology
(e.g.,BRCA1 and BRCA2), to define the causes
of inherited diseases, or to use genome-wide
mutation profiles for individualized medicine,
care, and treatment.
Personalized medicine
NSG vs Sanger sequencing
 Step by step overview
• DNA RNA extraction
• Nucleic Acid quantitation
• Enrichment
• Library preparation
• Library quantification
• Template preparation
• Sequencing
• Data analyses
Oncomine assay workflow
Nucleic acid extraction from tissue
Nucleic acid quantification
Step by step overview
RNA only – first step
Amplification
Amplicon pooling after PCR
Amplicon pooling after PCR
Amplicon pooling after PCR
Amplicon pooling after PCR
NGS steps
Attaching molecules to ION Speres
Template preparation
Sequence
Data analyses
Oncomine gene list
Mutation categories
 What is next?
• ClinVar
https://www.ncbi.nlm.nih.gov/clinvar/

• OncoKB
https://oncokb.org/

• MyCancerGenome
https://www.mycancergenome.org/
ALK mutation
ALK mutation
The importance and role of NGS
Cancer cases
Predictive markers
 Oncomine portfolio
• 2 x Oncomine Solid DNA panel → Colon and lung
• 1 x Oncomine Solid RNA panel → Lung cancers
• 1 x Oncomine Focus panel → Melanoma, GIST and Glioma
• 1 x Oncomine BRCA Research Assay → Ovarian, Breast and
Prostata
• 1 x Oncomine Comprehensive panels → Unknown cancer type
• 1 x Oncomine cfDNA lung/colon liquid biopsies → Lung and
colon patient follow-up
NGS report sample
Semiconductor sequencing technology Chip
sequencing
 Reading
• Clinical Diagnosis and Management of
Laboratory Methods by Richard A.
McPherson, MD, MSc and Matthew R. Pincus,
MD, PhD
• Pages 52-53 65-68, 1313-1315,
• Electronic book is uploaded in TMA.INI.GE