Application of PCR and qPCR in diagnostic medicine

Application of PCR and qPCR in diagnostic medicine
Asst. Prof. Dr. Thanusak Tatu
Division of Clinical Microscopy
Department of Medical Technology
Faculty of Associated Medical Sciences
Chiang Mai University
Polymerase Chain Reaction (PCR)
A biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment or sequence of interest of DNA, via enzymatic replication, without using a living organism (such as E. coli or yeast).
As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA
Can be extensively modified to perform a wide array of genetic manipulations
http://users.ugent.be/~avierstr/principles/pcrani.html
Animation for PCR
General types of PCR
Traditional PCR
Quantitative PCR
Traditional PCR
End-point detection
Size-based discrimination only
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/
DNA_and_RNA_Purification/Extract_N_Amp_Blood_PCR_Kit.html
Real-time PCR
In-process detection
Threshold cycle (CT)-detection
Dissociation curve analysis
http://molecular.roche.com/diagnostics/microbiology/products_microbiology_05.html
http://bmc.ub.uni-potsdam.de/1471-2172-6-5/F1.htm
Amplification plot
Standard curve
Traditional PCR in
diagnostic medicine
Mutation detection (known and unknown mutaions)
Detection of infectious organisms
Unknown mutations
PCR-SSCP (PCR-Single strand conformational polymorphism)
PCR-DGGE (PCR-Denaturing gradient gel electrophoresis)
PCR-RFLP haplotype analysis
PCR-SSCP
http://biotech.szbk.u-szeged.hu/CG_Jegyzet/02_ora/abrak/htmlhez/modszerek/SSCP.jpg
PCR-DGGE
http://biotech.szbk.u-szeged.hu/CG_Jegyzet/02_ora/abrak/htmlhez/modszerek/dgge1.jpg
PND for b-thalassemia using haplotype analysis of RFLP
e
Gg
Ag
yb
d
b
HincII
Hind III
Hinc II
Ava II
Bam HI
PCR-RFLP haplotype analysis
HincII-e
HindIII-Gg
HindIII-Ag
HincII-yb
HincII-3’yb
AvaII-b
BamHi-3’b

+
-
-
+
+
-
+
-
-
-
+
-
+
-
+
+
-
+
+
+
-
+
+
+
+
+
-
-
+
+
+
+
+
-
-
+
+
-
+
+
+
-
-
-
-
+
-
+
-
+
-
-
+
+
-
+
Father
Mother
Affected child
In utero baby
Diagnosis
Non-disease baby
Known mutations
Allele-specific PCR (ARMS, MS, Gap)
PCR with restriction digestion of amplified products
PCR with ASO-dot blot
PCR with RDB
ARMS-PCR for b-thalassemia
mutation
Agarose gel
ARMS- PCR for Hb E
Family 2
Family 2
1.Mother (- /-)
2.Brother (- /-)
3.Father (+/-)
4.Propositus (+ /-)
Negative control (- /-)
Positive control (+/-)
M
M
M
M
M
M
N
N
N
N
N
N
Marker
1
2
3
4
N
P
314 bp
200 bp
Marker;Ø174 RF DNA / Hae III Fragment
MS-PCR for b-thalassemia
mutation
Agarose gel
MS-PCR for CD17 (A-T)
of b-globin gene
1 2 3 4 5 M
1, 2, 3 : Negative
4, 5 : Heterozygote
190 bp
170 bp
Gap-PCR for a-thal 1
(For SEA type)
314 bp
Normal
188 bp
a-thal 1
Agarose gel
PCR for a -thal 1 (SEA type)
314 bp
188 bp

1. Father = Negative (aa/aa)
2. Mother = Heterozygous (--/aa)
3. Sister = Heterozygous (--/aa)
4. Daughter = Heterozygous (--/aa)
5. Positive control (--/aa)
M = f X 174 Hae III digest
1 2 3 4 5 M
Multiplex PCR for a-thalassemia
Chong SS, Boehm CD, Higgs DR, Cutting GR. Single-tube multiplex-PCR screen
for common deletional determinants of alpha-thalassemia. Blood. 2000 Jan 1;95(1):360-2.
PCR with restriction digestion of
amplified products
Mutation creating cutting site
665
445
220
C/C
T/T
Result of digestion of PCR products
1
2
3
4
5
6
7
8
N
P
Marker
665 bp
445 bp
220 bp
Family 1
1.Brother (+ /-)
2.Propositus (+ /-)
3.Father (- /-)
4.Mother (+/+)
Family 2
5.Mother (- /-)
6.Brother(+ /-)
7.Father (+/+)
8.Propositus (+ /-)
Negative control (- /-)
Positive control (+/+)
Marker;Ø174 RF DNA / Hae III Fragment
PCR with ASO-dot blot
PCR
Dot on Nylon membrane
Hybridization with
normal and mutant probes
Color development
Result
All negative
PCR with RDB
Oligo-probes
attached to membrane
PCR products hybridized
to membrane bound probes
Color development
Real-time PCR in
diagnostic medicine
Mutation detection
Quantitation of initial copy number of DNA or RNA
Mutation detection
For known mutations only
By dissociation curve or melting point analysis
Dissociation curve analysis for
b-hemoglobinopathies
Herrmann MG, Dobrowolski SF, Wittwer CT. Rapid beta-globin genotyping by multiplexing probe melting temperature and color. Clin Chem. 2000 Mar;46(3):425-8.
Dissociation curve analysis for
b-hemoglobinopathies
Herrmann MG, Dobrowolski SF, Wittwer CT. Rapid beta-globin genotyping by multiplexing probe melting temperature and color. Clin Chem. 2000 Mar;46(3):425-8.
Dissociation curve analysis for
a-thalassemia
Dissociation curve analysis for
a-thalassemia
Liu J, Yan M, Wang Z, Wang L, Zhou Y, Xiao B. Molecular diagnosis of alpha-thalassemia by
combining real-time PCR with SYBR Green1 and dissociation curve analysis. Transl Res. 2006 Jul;148(1):6-12.
Dissociation curve analysis for
a-thalassemia
Liu J, Yan M, Wang Z, Wang L, Zhou Y, Xiao B. Molecular diagnosis of alpha-thalassemia by
combining real-time PCR with SYBR Green1 and dissociation curve analysis. Transl Res. 2006 Jul;148(1):6-12.
Dissociation curve analysis for
a-thalassemia
Liu J, Yan M, Wang Z, Wang L, Zhou Y, Xiao B. Molecular diagnosis of alpha-thalassemia by
combining real-time PCR with SYBR Green1 and dissociation curve analysis. Transl Res. 2006 Jul;148(1):6-12.
Quantitation of copy number
By using amplification plot and standard curve
http://www.finnzymes.fi/realtime_qpcr/dynamo_sybr_green_qpcr_kit_incl_ROX_pass_ref_dye.html
Quantitation of copy number for viral load
Leung A Y H, Yuen K-Y and Kwong Y-L. Polyoma BK virus and haemorrhagic cystitis in haematopoietic stem cell transplantation: a changing paradigm (review). Bone Marrow Transplantation (2005) 36, 929–37.
Good Bye