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					QUANTIFYING RENIFORM
NEMATODES USING
QUANTITATIVE REAL TIME              PCR
MARKERS




India Brown
Research Experience For Undergraduates
Summer 2009
OBJECTIVE

   The objective of this research was to develop a
    technique for real-time identification and
    quantification of reniform nematodes from
    infected soil samples, and detect differences in
    reniform nematode quantities among various
    soils of different plants.
RENIFORM NEMATODE
 The reniform nematode is considered to be the
  most underrated nematode parasite of cotton
 It was first observed on the roots of cowpea in
  Hawaii.
 It is often overlooked because roots of infected
  plants do not become knotted, distorted, or
  abnormal
 It is responsible for major crop loss and is unique
  because the immature and mature female
  nematode parasitizes roots, and is able to
  reproduce and develop
GOSSYPIUM LONGICALYX
   Resistant to reniform nematode

   The mechanisms for resistance are not fully
    known

   The resistance has been shown to be genetic and
    can probably be transferred to upland cotton

   Extensive studies are underway to introgress this
    resistance into upland cotton to reduce crop
    losses due to the reniform nematode in the USA.
BACKGROUND ON THE TOMATO PLANT
   Tomato (Solanum lycopersicum M.) belongs to
    the Solanacea family that includes 300 species

   Wild tomatoes have a large genetic diversity:
    sources of useful traits in tomato breeding such
    as disease resistances, environmental tolerances

o   Tomato offers an accessible model system for
    reniform nematode resistance study
    o large number of germplasm or mutants collection
    o prolific seed production and easy cultivation in
      restricted space
    o early maturation (60-95 days).
PRIMERS

 A primer is a strand of nucleic acid that serves as
  a starting point for DNA replication.
 They are required because the enzymes involved
  in replication (DNA polymerases) can only add
  new nucleotides to an existing strand of DNA
 These primers are usually short with a length of
  about twenty bases
 Primer (RNV1_C_FR) was designed to selectively
  amplify portions of the reniform nematode
  genome.
POLYMERASE CHAIN REACTION(PCR)
   A technique to amplify a single or few copies of a
    piece of DNA across several orders of magnitude,
    generating millions or more copies of a particular
    DNA sequence

   The method relies on thermal cycling, consisting
    of cycles of repeated heating and cooling of the
    reaction for DNA melting and enzymatic
    replication of the DNA.
QUANTITATIVE REAL TIME PCR
 Used to amplify and simultaneously quantify a
  targeted DNA molecule.
 It enables both detection and quantification of a
  specific sequence in a DNA sample
 The procedure follows the general principle of
  polymerase chain reaction; its key feature is that
  the amplified DNA is quantified as it
  accumulates in the reaction in real time after
  each amplification cycle
MATERIALS AND METHODS
   DNA was extracted from infected soil samples
     ZR Soil Microbe DNA Kit
     0.25 g of soil sample : Gossypium longicalyx:F4(1),
      F4(2), F4(3), F4(4), G. arboreum: A2-87(1),A2-
      87(2),A2-87(3), A2-87(4), and S. lycopersicum cv.
      Microtom: MT(1), MT(2), MT(3), MT(4).
CONT.
   DNA Extraction from reniform nematode
    inoculum

       DNEasy Blood & Tissue Kit(50) (Qiagen, Valencia,
        CA, USA)

       Sample: RN inoculum with 2000 juveniles and

    o   Serial dilutions: 1X(2000 nematodes), 2X, 4X, 8X,
        16X, 32X, 64X, 128X
CONT.
Polymerase Chain Reaction (PCR) on DNA
 Reaction volume: 25 µl
     2 µl of extracted DNA,
     2.5 µl 5x Go Taq buffer (Promega, Madison, WI,
      USA.),
     2.0 µl MgCl2 (25 mM),
     0.5 µl dNTPs (10 mM),
     1.0 µl of primer (10 µm),
     0.2 ul of taq DNA polymerase (Promega, Madison,
      WI, USA.)
     and double distilled water
CONT.

      Peltier Thermal Cycler (MJ research, Inc.
      Watertown, MA, USA)

      Parameters:

  o1. 95 oC 5 minutes
  o2. 95 oC 30 seconds
  o3. 60 oC 30 seconds -0.5/cycle
  o4. 72 oC 45 seconds
  o5. Go to step 2. 4 times
  o6. 95 oC 30 seconds
  o7. 57 oC 30 seconds
  o8. 72 oC 45 seconds
  o9. Go to step 6. 34 times
  o10. 72 oC 5 minutes
  o11. Store at 4 oC forever
CONT.
   Agarose Gel Electrophoresis

   The quality of PCR products were checked by
    electrophoresis of 7 µl of PCR reaction in 1%
    agarose gel with stain, visualized and
    photographed under Ultraviolet light.

   The size of each PCR product was determined by
    comparison to a 100 bp DNA marker.
CONT.
   Real Time qPCR Using SYBR Green Master I
    (Roche applied Science).

   DNA samples was analyzed using the LightCycler
    480 instrument

   Reaction volume:20 µl
       1 µl of sample DNA,
       2.0 µl of 10mM forward and reverse primers (RNV1_C),
       10 µl of Sybr Green Master Mix
       7 µl of PCR grade H2O.
       Reactions were performed in LightCycler 480 Multiwell
        Plate 96 sealed with sealing foil.
 RESULTS




             200 bp

                        M 1 2 3 4+ve -ve
Figure 1. Gel picture showing the results of PCR on G. longicalyx (F4)

      M= 100 bp DNA

      +ve = Reniform nematode (RN) DNA

      -ve = Dnase free Water

      1, 2, 3, 4 = RN DNA from soil obtained from
       four G. longicalyx plants
RESULTS CONT.




 Figure 2. Graph showing the results from qPCR on G. longicalyx
                1X = 2000 nematodes      32X         -VE
                2X            F4_4
                 4X            64X
                 8X            F4_4
                 16X            128X
RESULTS CONT.




 Figure 3. Graph showing results from qPCR soil samples obtained from
 tomato samples
RESULTS CONT.



        200bp
                        M 1   2   4 8 16 32 64 128 +ve -ve
Figure 4. Gel picture showing results from PCR on Serial Dilutions
M = 100 bp DNA ladder (Fisher)
+ve = RN DNA
-ve = Dnase free Water
1,2,4-128 =1:2 (two-fold) serial dilutions of RN DNA

oSerial dilutions gives up the exact number of nematodes which is
the standard we use to compare the samples
CONCLUSIONS
THANKS
   Questions?

				
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