Docstoc

DNA isolation kits - PowerPoint

Document Sample
DNA isolation kits - PowerPoint Powered By Docstoc
					NucleoSpin® Plasma XS
NucleoSpin® Plasma XS
XS columns – new column design for specific applications

 New column design – why in general?
  - customer do more and more often start with very small samples
   (e.g. for expression profiling, single cell characterizations)
  - customer asked for:
   high recovery of RNA/DNA in very small elution volumes


 New column design – why for plasma?
 - DNA in plasma is of low concentration and highly fragmented, standard
 kits fail to give satisfying DNA recovery and concentration
 Standard columns – the problems:
 - dead volumes of standard mini kits are normally to
 high to allow an elution in less than 50µl
 - smaller columns would not fit in standard centrifuges

          What to do?
NucleoSpin® Plasma XS
Qiagen‘s solution – MinElute columns



                                     standard columns



                                     broader thrust ring,
                                     smaller menbrane diameter

                                      thin silica menbrane




          The result: elution in 20 - 50µl possible
          Customer still ask for less elution volume
NucleoSpin® Plasma XS
 New columns – the design of our columns


                                       standard NucleoSpin
                                       columns

                                       funnel shaped
                                       thrust ring

                                       small membrane
                                       diameter

                                       small elution
                                       volumes


                                  Xtra Small elution volume
                                  Xtra Sensitive colums
                                  Xtra high DNA concentration
NucleoSpin® Plasma XS




                        Simple and fast
                        procedure, in less than
                        30 min from plasma to
                        circulating DNA
NucleoSpin® Plasma XS
 Recovery of fragmented DNA


 Recovery rates from plasma spikes

                                                       DNA spikes of 50, 100, 150,
                                                       250, and 1000 bp were
                                                       mixed with a plasma
                                                       sample. DNA was
                                                       subsequently purified with
                                                       NucleoSpin® Plasma XS
                                                       and a kit of competitor Q in
                                                       parallel.

                                                       MACHEREY-NAGEL in-house
                                                       data




     Competitor Q completely failed to show recovery for fragments
     smaller than 100 bp and resulted in an overall weaker performance
     compared to NucleoSpin® Plasma XS (MN) which yielded
     significantly higher DNA concentration.
NucleoSpin® Plasma XS
 DNA yield with multiple column loadings
                  100                                                                  Five different plasma samples with a
                                                                                       different total DNA content were used
                                                                                       as sample for DNA purification.
                                                                                       Multiples of the standard lysate (240µl
                                                                                       plasma + 360µl binding buffer) were
                                                                                       loaded.
                  10                                                                   Yield significantly increases at least
                                                                                                Plasma 1
 DNA yield [ng]




                                                                                       over the first three loading steps, thus
                                                                                       the kit can be highly recommended for
                                                                                                Plasma 2
                                                                                       up to 740 µl of plasma,
                                                                                               Plasma 3
                                                                                       MACHEREY-NAGEL in-house data
                                                                                               Plasma 4
                   1
                                                                                       DNA yield increases from
                                                                                               Plasma 5
                                                                                       one to three column
                                                                                       loadings
                                                                                       (for plasma of low as well as
                                                                                       for plasma of high DNA
                  0,1
                                                                                       content)
                        0           1            2            3              4
                        Number of column loadings with 600 µl lysate
                                               480µl plasma +
                          240µl plasma +                              720µl plasma +
                                             720µl binding buffer
                        360µl binding buffer                        1080µl binding buffer
NucleoSpin® Plasma XS
 Elution profile - concentration and yield

                                              DNA recovery depends on the
                                              elution volume.
                                              Below a certain volume, DNA
                                              yield decreases significantly with
                                              an decreasing elution volume
                                              (due to the dead volume of the
                                              silica membrane).

                                              Qiagen´s MinElute columns
                                              require at least 20µl elution
                                              volume. Below this, yield (blue)
                                              decreases and DNA
                                              concentration (red) can not be
                                              increased (Q).

                                              NucleoSpin XS columns allow a
                                              significantly smaller elution
                                              volume, thus DNA concentration
                                              (red) can significantly be
      NucleoSpin Plasma XS allows small       increased (MN).
      elution volumes and results in highly
                                              MACHEREY-NAGEL in-house data
      concentrated DNA
NucleoSpin® Plasma XS
 Sales arguments


  Arguments                              Customer benefits
► silica membrane technology            …known for high reliability
► optimized column design               …reduced membrane area
► elution in only 5 - 20 μl             …gives you highly concentrated DNA
► high DNA concentration                …no need for subsequent concentration
► high purity                           …ready-to-use for real-time PCR
► high recovery of fragments >50bp      …ideal for fragmented circulating DNA
► fast preparation, easy handling       … convenient in use, requires less than 30 min
► standard protocol for 240 μl plasma   … ideal solution for your routine analysis
► multiple loading steps possible       … a solution even for up to ~720 µl of plasma




A solution for an existing problem, no other kit is existing in the market yet!
NucleoSpin® Plasma XS
 Application data




                    Analysis of cancer
NucleoSpin® Plasma XS
 Analysis of cancer

 Biopsy analysis:
 In case of cancer suspicion (e.g. tissue alteration), a common diagnostic
 tool is the extraction of a biopsy sample for subsequent histological and
 genetic analysis



                                    Risks and Intricacies?

                                    - injury, bleedings
                                    - infections
                                    - vascular and organ damage
                                    - spreading of cancer cells




                     None invasive alternatives requested!
                     How can NucleoSpin® Plasma XS help?
NucleoSpin® Plasma XS
 Analysis of cancer

 Increased amounts of circulating DNA in plasma have been found in disorders
 including cancer, autoimmune diseases, infections, certain fetal diseases,
 trauma, stroke, and others.


     Tumor cells           Lymphocytes destroy tumor cells      Degradation
    Cells affected                                           products circulate
                             Apoptosis deregulation           in blood plasma
    by disorders



                                                                    Blood draw /
                                                                    Plasma recovery




 - oncogenes                                                     Circulating DNA:
 - tumor suppr. genes           NucleoSpin® Plasma XS
                                                               - highly fragmented
 - microsat. alterations                                     - low in concentration
 - point mutations

                                                         None invasive , highly sensitive
NucleoSpin® Plasma XS
 Analysis of cancer

 NucleoSpin® Plasma XS is superior to competitor Midi kits - Better
 PCR signals from less volume of plasma

                                               Starting material: Plasma samples
                                               originating from rat EDTA blood
                                               PCR amplification: 165 bp fragment
                                               of the k-ras gene (often involved in
                                               cancer).
                                               Comparison: QIAamp DNA Blood
                                               Midi, NucleoSpin® Plasma XS
                                               QIAamp DNA Blood Midi, input 1000
                                               μl plasma, purification according to the
                                               user manual, modification: 100μl
                                               elution buffer were pre-heated to 56°C,
                                               5 min incubation on the membrane
                                               prior to elution, elution fraction
                                               concentrated to 30 μl, 2 μl of
                                               concentrated eluate used for PCR
                                               NucleoSpin® Plasma XS, input 240 μl
                                               plasma, purification according to the
                                               user manual without modification,
    NucleoSpin® Plasma XS shows an             elution with 20 μl, 2 μl of eluate used
    earlier PCR signal of 2.1 cycles even      for PCR
    though QIAamp DNA Blood Midi               Data kindly provided by Dolores C. García-
    represents a 2.7 fold PCR input            Olmo, PhD, Unidad de Investigación,
                                               Complejo Hospitalario Universitario de
    compared to NucleoSpin® Plasma XS.         Albacete, Albacete, Spain
NucleoSpin® Plasma XS
 Application data




                    Analysis of fetal DNA
NucleoSpin® Plasma XS
 Analysis of fetal DNA
 Today´s solution:
 Amniocentesis - amniotic fluid contains fetal cells for early DNA diagnosis


                                        When indicated?
                                        - pregnant women > 35 of age , higher risk of
                                          genetic mutations, risk of handicaps
                                        - genetic defects in the family
                                        - identification of developmental
                                          disorders
                                        - test of blood group incompatibility


                                        Risks and Intricacies?
                                        - injury of the child by injection needle
                                        - bleeding in the uterus
                                        - rate of abortion 0,5%!



            None invasive replacement of Amniocentesis requested!
                   How can NucleoSpin® Plasma XS help?
NucleoSpin® Plasma XS
 Analysis of fetal DNA
 Future´s Solution:
 Placental circulation connects maternal and fetal bloodstream


                                       Fetal and maternal circulations are
                                       separated by placental membranes. This
                                       barrier seems to be incomplete to
                                       cellular trafficking

                                       Molecular analysis of plasma DNA during
                                       human pregnancy has led to the
                                       discovery that maternal plasma contains
                                       both fetal and maternal DNA.




 Conclusions:
 Non invasive blood draw from pregnant woman can replace amniocentesis
 Robust and reliable nucleic acid purification kit required
 (especially for fragmented cell free DNA)
                                             NucleoSpin® Plasma XS
NucleoSpin® Plasma XS
 Analysis of fetal DNA

 NucleoSpin® Plasma XS is ideally suited for the detection of fetal DNA
 in maternal plasma

                                                   Starting material: Human maternal
                                                   plasma used for fetal DNA diagnostics
                                                   Duplex PCR amplification: RHD
                                                   fragment (Rhesus-gene) and an
                                                   Amelogenin fragment specific for the
                                                   male Y-chromosome, amplicons 150 –
                                                   180 bp in size.
                                                   Comparison: Roche MagNA Pure
                                                   Compact Nucleic Isolation Kit I - Large
                                                   Volume, NucleoSpin® Plasma XS.
                                                   Roche MagNA Pure Compact Nucleic
                                                   Isolation Kit I – Large Volume, input 1000
                                                   μl plasma, purification according to the
                                                   user manual, 10% of the eluate used for
                                                   PCR (i.e. DNA from approx. 100 μl
                                                   plasma) NucleoSpin® Plasma XS, input
                                                   240 μl plasma, purification according to
   The significantly better performance of         the user manual, 40% of the eluate used
                                                   for PCR (i.e. DNA from approx. 100 μl
   NucleoSpin® Plasma XS is demonstrated by        plasma)
   an earlier PCR signal of 2.6 cycles (RHD
   gene) and 2.1 cycles (Amelogenin)                 Data kindly provided by Dr.
   respectively.                                     Doescher, DRK Blutspendedienst
                                                     N.S.T.O.B.,Oldenburg, Germany
NucleoSpin® Plasma XS
 Customer testimonals


 We tested NucleoSpin® Plasma XS using human maternal plasma samples for
 subsequent fetal blood group genotyping (fetal RHD, RHE, and KEL typing).
 NucleoSpin® Plasma XS gave a 2.5 – 3 cycles earlier signal compared to the
 competitor kit we used so far. I like the easy handling, the short purification
 procedure, and the good results!

 Dr. Doescher, DRK Blutspendedienst NSTOB, Oldenburg, Germany




 We tested NucleoSpin® Plasma XS using rat plasma samples and are very
 satisfied.
 In plasma samples from healthy rats, we found significantly higher amounts of
 DNA compared to the competitor kit we used so far (PCR target: k-ras). In addition
 to this, less volume of plasma is sufficient (240 μl in contrast to 1 ml which we
 needed with the present kit) and the procedure is quicker!

 Dolores C. García-Olmo, PhD, Unidad de Investigación, Complejo Hospitalario Universitario de
 Albacete, Albacete, Spain
NucleoSpin® Plasma XS
 Summary




                   NucleoSpin® Plasma XS
        The only kit in the market that gives you highly
          concentrated circulating DNA from plasma

           - higher yields and recovery rates than competitors!
     - less plasma needed - a mini prep is often enough for analysis

                        - not available from Qiagen
                  - not only an alternative… a solution!

				
DOCUMENT INFO
Shared By:
Categories:
Stats:
views:85
posted:11/7/2010
language:English
pages:20