Dna Based Molecular Diagnosis(CMH)Final (1).pptx

Abu Sufian
DNA Analyst
1st molecular Diagnostic Lab
in Bangladesh
DNA/RNA Based Molecular Diagnosis

Diagnosis based on the
detection of DNA & RNA.
Methods:
1. PCR
2. Real-time PCR
3. Sequencing
Molecular Diagnostics

Genetic Materials (DNA & RNA)
First Molecular Diagnostic Lab in Bangladesh

DNA
Deoxyribonucleic acid (DNA) is a molecule that carries the genetic
instructions used in the growth, development, functioning
and reproduction of all known living organisms and many viruses.
A T G C

DNA Located inside human body
Humans are multi-
cellular organism
There are ~ 100 trillion cells in an
adult human body
Humans have 23 pairs
of chromosome
Chromosome
DNA Double Helix

RNA
 Ribonucleic acid (RNA) is a nucleic acid present in all living cells.
 Its principal role is to act as a messenger carrying instructions from DNA for
controlling the synthesis of proteins.
 In some viruses RNA carries the genetic information.

Codon Change
(GAG > GTG)

 Molecular virology
 Detection of viral pathogens
 Quantitation of viral copy number
 Genotyping of viral strains
▪ HBV, HCV, HIV, HPV, Influenza (H1N1, H5N1) etc.
 Molecular bacteriology
 Detection of bacterial pathogens
▪ MTB, Salmonella, Shigella , etc.
 Molecular genetics
 Diagnosis of genetic disease
▪ -thalassemia, Factor V Leiden, DMD, Fragile X syndrome

 Molecular oncology
 Detection of the expression of an oncogene
▪ N-myc, BRCA1/BRCA2 , HER2/neu
 Quantitative expression of fusion transcripts
▪ BCR-ABL t(9:22), PML-RARA t(15:17), TEL-AML1
 Molecular cytogenetics
 Karyo typing
 Detection of chromosomal abnormalities
▪ Tisomy 21 (Down’s syndrome
▪ Trisomy 18 (Edward syndrome)
▪ Trisomy 13 (Patau syndrome)
▪ Turner syndrome (XO)
▪ Kleinfelter syndrome (47, XXY)
 Transplantation genetics
 Tissue typing
▪ HLA-A, B, DQ & DR Typing

 Personalized medicine /Pharmacogenomics
 Drug efficacy
▪ Warfarin sensitivity (CYP2C9, VKORC1)
▪ Beta blocker metabolism (CYP2D6 genotyping)
 Detection of drug susceptibility
▪ Herceptin
▪ Tyrosine kinase inhibitor
 GMO Plant screening
 GMO Soya
 Terminator Nos corn
 GMO Maize
 GMO Rice
 Drug resistance
 MTB (Rifampicine resistance)
 MRSA (Methicilline resistance Staphylococcus aureus)
 HBV drug resistance

Sensitivity: Amplification methodologies are particularly useful in increasing the
sensitivity of a methodology and useful in the identification of target
molecules of interest that are only present in low concentrations.
Specificity: Molecular methods minimize false positive test results by targeting the
specific molecule of interest.
Turn Around
Time:
In comparison with standard traditional culture methods, molecular
methodologies usually offer better turn around times from receipt to
result reporting.
Application: Broader application can be found with molecular methodologies such as
infectious diseases, genetic testing, forensics, drug resistance, and tumor
marker detection and monitoring.

Technique Description Comment
Hybridization Single stranded DNA probes binds directly
to target of interest
• Since the target DNA is not
amplified, the technique can be less
sensitive.
PCR DNA or RNA is amplified to detect the
presence of a pathogen or host gene of
interest
• Always need a post-PCR
processing step which is usually
time consuming.
Real Time PCR DNA or RNA is amplified and detected at
real time as the amplicons are generated
• Sensitive than conventional PCR
• No post-PCR processing step
• Costly compared to earlier
methods
Sequencing The specific nucleotide sequence is
determined for an amplified piece of DNA
or the full genome of an organism
• Provides more information
• Expensive lab & equipment
• Require skilled manpower
Basic Molecular Diagnostic Techniques

Extraction
Pure DNA
DNA in Cell

PCR + Electrophoresis
Blood
Amniotic fluid
CVS
No need of cell culture
DNA/RNA
Extraction
PCR
amplification of
chromosome
specific STR
sequences
Capillary electrophoresis
Electrophoresis

Real Time PCR
DNA/RNA
Extraction
PCR amplification
and detection
Sample

DNA Evidence Genomic DNA
PCR Amplification of
multiple STR loci
PCR Products
ABI 3500 Genetic
Analyzer
Sequencing

DNA Evidence Genomic DNA
PCR Amplification of
multiple STR loci PCR Products
ABI 3500 Genetic
Analyzer
Fragment Analysis

Types of chromosomal abnormalities
• Numerical
– Aneuploidy
– Polyploidy
• Structural
– Translocations: Chromosomal segment is moved from one position
to another
– Deletions – loss of a chromosomal segment
– Duplication - repeat of a chromosomal segment
– Inversion – reversal of a chromosomal segment
• Single gene defect
• Mutation (e.g. Cystic fibrosis, sickle cell anemia)
• Repeat expansion (e.g. Fragile X syndrome- CGG repeat expansion)

Abnormality Chromosome
involved
Frequency
Down syndrome Trisomy 21 ~1 in 800
Edward syndrome Trisomy 18 ~ 1 in 3,000
Patau syndrome Trisomy 13 ~ 1 in 10,00
Autosomal aneuploidies:

Sex chromosome aneuploidies:
Abnormality Chromosome
involved
Frequency
Turner syndrome 45, X ~1 in 2500
Klinefelter syndrome 47, XXY ~ 1 in 1200
Triple X syndrome 47, XXX ~ 1 in 1500
Jacob syndrome 47, XYY ~ 1 in 1000

Chromosomal Aneuploidy Check
Chromosome 13
Chromosome 18
Chromosome 21
Y Chromosome specific marker
Gender marker
X chromosome counting marker
X chromosome marker (polymorphic)

Chromosomal Aneuploidy Check

Y-Chromosome Microdeletions
 Responsible for azoospermia, severe
oligospermia or male infertility.
 Microdeletion of either the entire AZFa or
AZFb regions of the Y chromosome shows
poor prognosis for sperm retrieval.
 On the other hand, majority of men with AZFc
deletion have sperm retrieval successfully for
IVF or ICSI (Intracytoplasmic sperm infection).

Q
Q
Q
sY255
sY131
sY90 sY81 sY625 sY127 sY134
sY86
sY84 sY254 M259 ZFY/ZFX Sy14
sY255
sY131
sY90 sY81 sY625 sY127 sY134
sY86
sY84 sY254 M259 ZFY/ZFX Sy14
sY255
sY131
sY90 sY81 sY625 sY127 sY134 sY254 M259 ZFY/ZFX Sy14
sY84
sY157
sY157
sY157
sY86
AZFc
AZFc
AZFc
AZFa
AZFa
AZFa
AZFa
AZFb AZFb
AZFb SRY
Deleted
Deleted
Deleted
Non specific Used as internal
control of PCR
Used as internal
control of PCR
Used as internal
control of PCR
Unique sequence present on
both X and Y chromosome

AML Screening Panel
Detects 11 fusion transcripts with 34 transcript variants
Fusion gene Incidence
Chromosomal
aberrations
Variants Result
AML 1-ETO AML t(8; 21) (q22; q22) - Negative
BCR-ABL CML (~85%), AML (~2%) t(9;22) (q34; q11) e1a3 Negative
e1a2 Negative
b3a2 Negative
b3a3 Negative
b2a2 Negative
b2a3 Negative
CALM-AF10 ALL/AML t(10;11) (q13; q14) AF10_240-CALM_1987 Negative
AF10_240-CALM_2092 Negative
CBFB-MYH11 AML Inv(16) (q13; q22) Type A Negative
Type B Negative
Type C Negative
Type D Negative
Type E Negative
Type F Negative
Type G Negative
Type H Negative
Type I Negative
Type J Negative
DEK-CAN AML t(6; 9) (q23; q34) - Negative
MLL-AF6 ALL/AML/MDS t(6; 11) (q27; q23) - Negative
MLL-AF9 AML t(9; 11) (q22; q23) 6A_S(THP-1) Negative
6A_L(THP-1 Negative
7A_(10A) Negative
8A_(MM6) Negative
6B_(9B) Negative
MLL-ELL AML/ALL t(11; 19) (q23; q13.1) e10e2 Negative
e10e3 Negative
MLL-PTD AML/ALL Partial tandem
duplication
e9e3 Negative
e10e3 Negative
e11e3 Negative
NPM1-MLF1 AML t(3; 5) (q25.1; q34) - Negative
PML-RARA AML/APL t(15; 17) (q22; q21) bcr1 (PR-L) Positive
bcr2 (PR-V) Negative
bcr3 (PR-S) Negative

Result & Report

FLT3 (ITD & D835)
FLT3 mutations occur in approximately 30% of AML patients and confer
a poor prognosis.

FLT3 (ITD & D835)
WT (Control)
WT (Sample)
Mut (Control)

Chimerism Testing Using STR

Chimerism Testing Using STR:
Engraftment Analysis

Alpha Thalassemia
Mutations
screened:
-α3.7,-α4.2,-(α)20.5,--SEA,--MED,--FIL,--THAI

Beta Thalassemia
In Beta Globin Gene:
 911 Hemoglobin variants .
 Some of them are pathogenic.
 Some can make β0 or β+ thalassemia.

Beta Thalassemia
Father:
Het: IVS-1-5 (G>C)
Mother:
Het: IVS-1-5 (G>C)
AF:
Homo: IVS-1-5 (G>C)

Warfarin Sensitivity Genotyping
CYP2C9 {CYC2C9*2 (C/T), & CYP2C9*3 (A/C)}
Genotype Number of Metabolism state
SNP 430, 1075 variant alleles Warfarin
(metaboliser)
*1/*1 0 fast
*1/*2
1 intermediary
*1/*3
*2/*2
2 slow
*2/*3
*3/*3
VKORC1
Genotype Number of Sensitivity
SNP 1639 variant alleles to Warfarin
A/A homozygote 2 high
G/A heterozygote 1 intermediary
G/G wild type 0 low
VKORC1 CYP2C9
*1/*1
Normal/Extensive
metabolizer
*1/*2
Intermediate
metabolizer
*1/*3
Slow
metabolizer
*2/*2
Slow
metabolizer
*2/*3
Slow
metabolizer
*3/*3
Very slow metabolizer
GG
Low sensitivity
5.0 – 7.0 mg 5.0 – 7.0 mg 3.0 – 4.0 mg 3.0 – 4.0 mg 3.0 – 4.0 mg 0.5 – 2.0 mg
AG
Medium sensitivity
5.0 – 7.0 mg 3.0 – 4.0 mg 3.0 – 4.0 mg 3.0 – 4.0 mg 0.5 – 2.0 mg 0.5 – 2.0 mg
AA
High sensitivity
3.0 – 4.0 mg 3.0 – 4.0 mg 0.5 – 2.0 mg 0.5 – 2.0 mg 0.5 – 2.0 mg 0.5 – 2.0 mg
RESULT
CYP2C9 Genotype : *1/*1 (Normal/Extensive metabolizer)
VKORC1 Genotype : GG (Low sensitivity to warfarin)
Suggested dose : 5.0 – 7.0 mg

SL Test Name
1 AML Screening Panel (11 Markers Including BCR-ABL,
PML-RARA)
2 BCR-ABL(Qualitative & Quantitative)
3 PML-RARA (Qualitative)
4 Immunophenotyping for Leukamia (Flow Cytometry)
5 FLT3 Prognostic Marker for AML
6 α-Thalassemia Mutation Detection
7 β-Thalassemia Mutation Detection
8 Hb Electrophoresis
9 Bone Marrow Study
10 Chimerism
11 HLA-A typing (DNA based)
12 HLA-B typing (DNA based)
13 HLA-DR typing (DNA based)
14 T Cell/B Cell Cross match
15 HLA-B27 DNA
16 HBV DNA (Quantitative & Genotyping)
SL Test Name
17 HCV RNA (Quantitative& Genotyping
18 Mycobacterium Tuberculosis (MTB) DNA
19 Human Papilloma virus DNA
20 HLA- B*1502 DNA
21 JAK- 2 Mutation Exon 14
22 Warfarin Sensitivity Genotyping Test
23 Clopidogrel PGx Test
24 Achondroplasia
25 Y-Chromosome Microdeletions (Male Infertility)
26 Chromosomal Aneuploidy Check (Trisomy 13, 18, 21, X
and Y) by QF-PCR. (Karyotyping Full Panel)
27 Down's Syndrome (Trisomy-21)
28 Sex Chromosome
29 Triple Test (AFP, β-hcG, uE3) with Trisomy Risk
Assessment (TRA)
30 DNA Profiling
31 DNA Paternity/Maternity test
32 DNA Relationship Test for Transplantation
33 DNA Relationship Test for Immigration
Our Services

Extraction Lab
Analysis Lab
PCR Lab
DNA Solution Lab

Expert Panel
Prof. Dr. Sharif Akhtaruzzaman
Dept. of Genetics Engineering and
Biotechnology, University of Dhaka
Consultant
DNA Solution Ltd.
Dr. Abdul Khaleque
Dept. of Biochemistry & Biotechnology
North South University
Consultant
DNA Solution Ltd.
Dr. Kazi Nadim Hasan
Dept. of Biochemistry & Biotechnology
North South University
Consultant
DNA Solution Ltd.
• Experienced & Qualified Laboratory Personnel

Reception

Working Station

General Lab

Extraction Lab

PCR Lab

Analysis Lab

THANK YOU

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