Application of PCR and qPCR in diagnostic medicine

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					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
 Animation for PCR




http://users.ugent.be/~avierstr/principles/pcrani.html
General types of PCR

 • Traditional PCR
 • Quantitative PCR
                 Traditional PCR




Size-based discrimination only                                     End-point detection
          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                                    Dissociation curve analysis




             In-process detection
                                                                 Amplification plot   Standard curve

  http://bmc.ub.uni-potsdam.de/1471-2172-6-5/F1.htm            Threshold cycle (CT)-detection
http://molecular.roche.com/diagnostics/microbiology/products_microbiology_05.html
 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
   Diagnosis                 PCR-RFLP haplotype analysis


Non-disease baby             Father            Mother

                                              Affected child           In utero baby

               HincII-e      +        -   +          +   +     +   -      +
               HindIII-Gg    +        -   +          -   +     +   -      -
               HindIII-Ag    +        -   -          -   +     -   -      -
               HincII-yb     +        +   +          +   +     +   +      +
               HincII-3’yb   +        -   +          +   +     +   -      +
               AvaII-b       -        +   +          -   -     +   +      -
               BamHi-3’b     -        -   -          +   -     -   -      +
        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 (+/-)
314 bp                                               4.Propositus (+ /-)
                                                     Negative control (- /-)
                                                     Positive control (+/-)
200 bp


         M N M N M N M            N M N M N      Marker

          1     2      3      4      N     P
         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



                        190 bp
                        170 bp
                                 1, 2, 3 : Negative
                                 4, 5 : Heterozygote
             Gap-PCR for a-thal 1
              (For SEA type)


Normal
                        314 bp

                                    Agarose gel
  a-thal 1                188 bp
 PCR for a -thal 1 (SEA type)

           1 2 3 4 5M
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
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.
    Mutation creating cutting site




                   665                 C/C

      445                        220   T/T




PCR with restriction digestion of
     amplified products
         Result of digestion of PCR products
                                                      Family 1
                                                      1.Brother (+ /-)
                                                      2.Propositus (+ /-)
665 bp
                                                      3.Father (- /-)
                                                      4.Mother (+/+)
445 bp                                                Family 2
                                                      5.Mother (- /-)
220 bp
                                                      6.Brother(+ /-)
                                                      7.Father (+/+)
                                                      8.Propositus (+ /-)
                                                      Negative control (- /-)
          1   2   3   4   5 6 7 8     N P Marker      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.
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