Note: Descriptions are shown in the official language in which they were submitted.
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1
A METHOD OF MODIFYING A MACROMOLECULE WITHOUT PRIOR
EXTRACTION FROM A SAMPLE
BACKGROUND OF THE INVENTION
The present invention encompasses a method of modifying a
macromolecule without prior extraction from a sample by converting the
macromolecule in the sample with a chemical, removing or converting chemical
intermediates, if necessary; and purifying the resulting modified
macromolecule.
One method used by vertebrates and higher plants to regulation gene
expression is the methylation of cytosines found in CpG islands located in
promoter regions of various genes. In order to study this method of gene
regulation, techniques were developed to discriminate methylated cytosines
from unmethylated cytosines. One method is to chemically treat DNA in such a
way that the cytosines are converted to uracils while 5-methyl-cytosines are
not
significantly converted. Frommer et al. (1992). A systematic investigation ori
the critical parameters of the modification procedure has also been made.
Grunau et al. (2001). The treated DNA may be used as template for
methylation specific PCR (MSP). DNA methylation and methods related
thereto are discussed for instance in US patent publication nurnbers
20020197639,20030022215,20030032026,20030082600,20030087258,
20030096289,20030129620,20030148290,20030157510,20030170684,
20030215842,20030224040,20030232351,20040023279,20040038245,
20040048275,20040072197,20040086944,20040101843,20040115663,
20040132048,20040137474,20040146866,20040146868,20040152080,
20040171118,20040203048,20040241704,20040248090,20040248120,
20040265814,20050009059,20050019762,20050026183,20050053937,
20050064428,20050069879,20050079527,20050089870,20050130172,
20050153296, 20050196792, 20050208491, 20050208538, 20050214812,
20050233340, 20050239101, 20050260630, 20050266458, 20050287553 and
US patent numbers 5786146, 6214556, 6251594, 6331393 and 6335165.
DNA modification kits are commercially available, they convert purified
genomic DNA with unmethylated cytosines into genomic lacking unmethylated
cytosines but with additional uracils. The treatment is a two-step chemical
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process consisting a deamination reaction facilitated. by bisulfite and a
desulfonation step facilitated by sodium hydroxide: Typically the deamination
reaction is performed as a liquid and is terminated by incubation on ice
followed
by adding column binding buffer. Following solid phase binding and washing
the DNA is eluted and the desulfonation reaction is performed in a liquid.
Adding ethanol terminates the reaction and the modified DNA is cleaned up.by
precipitation. However, both commercially available kits (Zymo and Chemicon)
perform the desulfonation reaction while the DNA is bound on the column and
washing the column terminates the reaction. The treated DNA is eluted from
the column ready for MSP assay. The modification is tedious and has many
steps that cause yield loss and increase operator er"ror. All of the available
modification procedures begin with purified genomic DNA, which is a tedious
process that also has many steps that cause yield loss and increase operator
error.
SUMMARY OF THE INVENTION
The present invention encompasses a method of modifying a.
macromolecule without prior extraction from a sample by converting the
macromolecule in the sample with a chemical, removing or converting chemical
intermediates, if necessary; and purifying the resulting modified
macromolecule.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1: Shows that DNA modification in without isolation from a biological
sample is equivalent to such modification after isolation.
DETAILED DESCRIPTION
The present invention encompasses a method of modifying a macromolecule
without prior extraction from a sample by converting the macromolecule in the
sample with a chemical, removing or converting chemical intermediates, if
necessary; and purifying the resulting modified macromolecule.
The macromolecules can be any known in the art including, without
30. limitation, DNA, RNA, cellular metabolites, lipids, carbohydrates and
proteins.
The DNA can be any known in the art including, without limitation, viral,
nucleic,
mitochondrial, plastid, bacterial and synthetic. The RNA can be any known in
the art including, without limitation, rtRNA, tRNA, miRNA, rRNA and mRNA.
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The cellular metabolite can be any known in the art including, without
limitation,
those produced by a metaboiic cycle or enzymatic effects. The lipid can be any
known in the art including, without limitation, liposomes, cell membrane
lipids,
intracellular membrane lipids and extracellular lipids. 'The carbohydrate can
be
any known in the art including, without limitation, protein-bound
carbohydrates
and nucleic acid-bound carbohydrates. 'The protein can be any known in the
art including, without limitation, intracelluiar and extracellular.
The modification is can be any known in the art including bisutfite and
biotinyfation of DNA or RNA, fluorination and methylation of RNA, heating,
liposome formation, micelle formation, uni-layer formation and bilayer
formation
of lipids, oxidation, de-oxidation; amination and de-amination of
carbohydrates
and phosphorylation, dephosphorylation, methylation, biotinylation, amination,
deamination, glycosylation and deglycosylation of proteins. 20070148670;
Chuang et al. (2007); Emmerechts et al. (2007); Frommer et al. (1992); Grunau
et al. (2001); Hurd et al. (2007); Jin et al. (2007); Oakeley (1999); Rathi et
al.
(2003); Rein et al. (1998); Sambrook et al. (2000); Wu et al. (2007).
The sample can be any known in the art including, without limitation,
tissue, body fluid, a biopsy sample, and preserved tissue. The tissue can be
any known in the art including, without limitation, whole organs, dissected
organs, epithelium, neural, gastrointestinal, muscle, cardiac, mucosal and
endothelium. The body fluid can be any known in the art including, without
limitation, whole blood, plasma, urine, saliva, vitreous and serum. The.biopsy
sample can be any known in the art including, without limitation, fine needle
aspirate, tissue section and skin sample. The preserved tissue can be any
known in the art including, without limitation, fresh frozen, paraffin
embedded
and preserved in a preservation reagent. The preservation reagent can be any
known in the art including, without limitation, formalin, RNAfater and
dimethylsulfoxide.
The purification can be any known in the art including, without limitation,
particle-based, precipitation, centrifugation, electrophoretic and charge
switch.
The particle-based purification can be any known in the art including, without
limitation, affinity, sizing and magnetic. The sizing particle can be any
known in
the art including, without limitation, silica-based and diatomaceous earth.
The
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electrophoretic separation purification can be any known in the art including,
without limitation, by size and/or charge. The electrophoretic separation
purification can be any known in the art including, without limitation, by a
gel
formed of low molecular weight polymers and/or capillary.
The present invention provides a rapid and efficient method for obtaining
bisulfite modified DNA. The method described herein effectively eliminates
numerous steps of the previous methods thus reducing possible error while
producing superior results. In addition considerable time savings of four to
five
hours are also realized.
The present invention provides a method of extracting and modifying
DNA by obtaining a DNA sample; incubating the sample with an amount of a
bisulfite and for a time and under conditions sufficient to convert at least
ninety-five percent of the non-methylated cytosine residues in the DNA to
uracil
resides; binding the DNA in the sample to a column; washing the bound DNA to
remove contaminants; incubating the column-bound DNA with a desulfonation
reagent for a time and under conditions sufficient for desulfonation to occur;
washing the bound DNA to remove the desulfonation reagent; and eluting the
bisulfite modified DNA from the column.
The DNA can be at a concentration of from about 0.01 to about 30 pg
and can be obtained by any method known in the art and can be purified DNA
or DNA obtained directly from a cell lysate. For instance, the cell lysate can
be
formed from any suitable tissue by any method known in the art and directly
treated with a bisulfite reagent. Cell lysis can be by for instance,
proteinase
and/or high salt concentration and/or detergent, sonication, freeze-thaw
treatment or mechanical disruption. Any cell sample is suitable for use herein
and can be obtained from tissue, body fluid, biopsy sample or preserved
tissue.
The bisulfite reagent can be any known in the art, including, without
limitation, sodium bisulfite or meta bisulfite. Other reagents are discussed
for
instance in US patent publications 20050089898, 20050095623 and
20050153308.
The incubation conditions of step b are about 1-16 hours at 50-95 C with
or without Thermocycling. Thermocycling can be for instance 3 hours at 70 C,
1 hour at 90 C or cycling between 50 C and 95 C.
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The column can be any known in the art, preferably, it is silica-based or
diatomaceous earth. The desulfonation can be by any method known in the art
and is preferably performed with sodium hydroxide and an alcohol. Preferably,
the alcohol is isopropanol or ethanol. More preferably, when the colurnn is
5 silica-based, the alcohol is ethanol and when the column is diatomaceous
earth, the alcohol is isopropanol. The desulfonation preferably occurs from
about 0-30, preferably about 5-15 and more preferably about 15 minutes at
about 0 C to about 50 C. Preferably, the temperature is about room
temperature.
The modified macromolecule can be eluted by any method known in the
art, including, without limitation with water or a suitable buffer.
The following examples are provided to illustrate but not limit the claimed
invention. All references cited herein are hereby incorporated herein by
reference.
Example I
Rapid bisulfite modification of DNA from a cell lysate obtained from fresh
frozen
paraffin embedded tissue
Microfuge tube containing cell culture pellet in PBS
= Add 100 - l DNA extraction buffer (75mM NaCl, 25mM EDTA, and 0.5%
Tween 20), incubate 56 C for 10minutes.
= Proceed with 3M NaOH addition described below
Or a microfuge tube containing (up to) 5 10 micron FFPE block slices.
The FFPE slices require deparaffination:
= Add 1 ml of Xylene to the tube and mix by inverting several times
: Incubate at RT for 5 min and centrifuge at full speed for 5minutes at RT
= Remove the supernatant without removing any pellet
= Repeat the I mt Xylene extraction
= Add 1 ml EtOH (100%) to the pellet and gently mix by inverting
= Centrifuge full speed for 5 minutes at RT
= Carefully remove the EtOH by pipetting without removing any of the
pellet
= Repeat the EtOH wash
= Dry the pellet in a 37 C heat block with the microfuge tubes open
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Extraction/modification procedure for FFPE samples
= Add 90 N1 extraction buffer (10mM Tris, pH 8.0, 150mM NaCI, 2mM
EDTA, and 0.5% SDS) and 10 pl proteinase K and incubate overnight at
56 C
= After incubation the extract should be clear, if not add 10 p proteinase K
and incubate for 1 hr
= Repeat until lysis is clear and proceed with 3M NaOH addition described
below.
= Add 1/10 volume 3M NaOH and incubate 56 C for 10minutes.
= Add 2 volumes of a saturated solution of sodium bisulfite pH 5.0 with
10mM hydroquinone, incubate at 70 C for 3 hours in the dark.
= Stop the reaction'by incubating on ice for 10 minutes. Add 0_5 volumes
of Isopropanol and gently vortex or pipette up and down.
== Add the sample to a Qiagen DNA purification column (QiaAmp DNA
purification kit), spin I min and empty waste tube;
= Add 500 NI AW1 wash buffer, spin 1 min and empty waste tube;
= Add 500 Ul AW2 wash buffer, spin 1 min and empty waste tube;
= Add 200 pl desulfonation buffer (300 mM NaOH, 80% Isopropanol),
incubate 10 min room temperature, spin 1 min and empty waste tube;
= Add 500 NI AW2 wash buffer, spin 1 min, empty waste tube and spin
1 min,
= Add 500 lal AW2 wash buffer, spin 1 min, empty waste tube and spin
1 min,
= Elute with 50 NI TE, spin 1 min into new 1.5 ml microfuge tube and store
at -20 C
The efficiency of the procedure is assayed using quantitative PCR and
(3-Actin using GSTPI as markers. The (3-Actin promoter is not methylated and
the marker is designed to serve as a control for the modification procedure.
The Ct value produced by this marker is reflective of the number of genome
equivalents added to the assay. Whereas the GSTP1 promoter is methylated
epigenetically and may be reflective of a cancerous state. Therefore the
GHSTP1 marker Ct value is more variable and usually greater than the ¾-Actin
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Ct value. The Prostate FFPE blocks were obtained from Asterand. The
"Zymo" treatment refers commercially available DNA modification kit sold as EZ
DNA Methylation Kit from Zymo Research. A 2 Step procedure refers to two
separate procedures that use a DNA purification kit (Qiagen QiaAmp mini
DBNA purification kit) and a DNA modification kit such as the Zymo kit. The
Zymo 1 step procedure is identical to the ID procedure until the 3M NaOH step
where the Zymo DNA modification procedure is followed replacing the 3M
NaOH with M-Dilution buffer.
The results shown in Table 1 using cell culture pellets indicate that the 1
Step method produces lower Ct values and thus superior results when
compared to the Qiagen/Zymo 2 Step procedure. Slatistically, a paired T test
indicates the methods, using the R-Actin marker, are significantly different
from
each other with a P value of 0.00006 while the GSTP1 marker had a P value of
0.0001. The results shown in Table 2 indicate that ID 1 step produces lower Ct
values when compared to the Zymo 1 Step method with the P-Actin marker
produces a P value of 0.02 and the GSTPI marker results in a P value of 0.003
However, the results shown in Table 1 using prostate FFPE blocks
indicate the opposite results from the cell cultures with the Zymo 2 Step
producing lower Ct values then the 1 step method. The (i-Actin marker results
indicate that the methods are significantly different with a P value of 0.021
while
the GSTP1 results suggest that they maybe different with a P value of 0.054.
The different results suggested that the extraction buffer for cell culture
might
not be sufficient to lysis tissue blocks. Table 4 results compares the ID 1
Step
and the Zymo 1 Step methods using a more aggressive extraction buffer
switching the Tween to SDS while using prostate tissue blocks. The ID 1 Step
method once again shows superior results suggesting that the failure to
produce superior results in Table 3 was due to the extraction buffer. The R-
Actin marker results indicates that the methods are significantly different
with a
P value of 0.0008 while the GSTP1 results suggest that they maybe different
with a P value of 0.056. Since the QPCR assay method only has 40 cycles,
once an assay fails to produce Ct's then it is not significant to compare with
assays that produce Ct's. If the two samples that had assays that failed then
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the P values would be 0.0025, which indicates the methods using the GSTP1
marker are significantly different.
Table 1: Results comparing the 1 Step procedure to the Qiagen/Zymo (Zymo) 2
Step procedure
Cell Culture LnCAP 1 x 10 Cell eltets, 2 pellets tested in du Iicate
Treatment Procedure Marker Ct SD P value
ID 1 Step P-Actin . 26.94 0.24 0.00006
Z mo 2 Step P-Actin 27.80 0.29
ID 1 Step GSTPI 27.66 0.20 0.0001
Z mo 2 Step GSTP1 28.54 0.23
Table 2: Results comparing the 1 Step procedure to the Zymo 1 Step
procedure
-
Cell Culture LnCAP 1 x 10 Cell eliets, 2 pellets tested in du licate
Treatment Procedure Marker Ct SD P value
ID 1 Ste P-Actin 27.60 0.18 0.020
Zymo 1 Step P-Actin 29.90 0.08
ID I Ste p GSTP1 28.48 0.07 0.00.3
Zymo 1 Step GSTP1 30.87 0.18
Table 3: Results comparing the 1 Step procedure to the Qiagen/Zymo (Zymo) 2
Ste procedure
Prostate FFPE Blocks Using The Cell Culture Extraction Buffer
Treatment Tissue Procedure Average SD Average = SD
Block -Actin Ct GSTP1 Ct
ID 1 1 Step 30.68 0.08 32.64 0.27
ID 2 1 Step 33.84 0.22 37.20 = 0.33
ID 3 1 Step 31.26 0.04 35.19 0.16
ID 4 1 Step 34.21 0.03 38.57 0.08
Zymo 1 2 Step 29.68 0.19 33.40 0.07
Zymo 2 2 Step 28.72 0:04 32.87 0.19
Z mo 3 2 Step 27.69 0.01 32.11 0.22
Zymo 4 2 Step 27.62 0.04 33.65 0.25
P value 0.021 00.054 -
Table 4: Results comparing the 1 Step procedure to the Zymo 1 Step
procedure using Prostate FFPE blocks.
Prostate FFPE Blocks Usin The FFPE Extraction Buffer
Treatment Tissue Block P-Actin P-Actin GSTPI GSTP1
Ct SD Ct SD
Z rno 12 35.16 0.10 40 0.00
ID 12 31.21 0.04 40 0.00
Zymo 16 34.41 0.12 35.76 0.79
ID = 16 30.23 0.33 31.98 0.25
Zymo 18 35.69 0.13 40.00 0.00
ID 18 32.63 0.16 39.27 1.03
Zymo 20 33.08 = 0.17 36.07 0.05
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ID 20 28.29 0.03 32.34 0.19
Control Plasmid 23.15 0.79 22.86 0.34
P value 00.0008 00.056
Example 2
Bisulfite treatment of DNA obtained from urine
The purpose of this experiment was to compare the DEM kit versus the
Zymo EZ Modification kit on purified DNA obtained from LnCAP cells and urine.
Ten normalized random samples were divided between the two kits
- 5 Samples per Kit (DEM and Zymo)
= PCR performed in duplicate using Fast Start Taq (GSTP1, R-Actin, and APC)
ANOVA analysis indicates a P value of 0.663 and a Paired T-Test
indicates that there is no statistical difference between DEM and Zymo for
GSTP1.
ANOVA analysis indicates a P value of 0.008 and a Paired T-Test
indicates that there is a statistical difference between DEM and Zymo for B-
Actin.
= The results indicate that Zymo and the DEM kit are equivalent for GSTPI
= The DEM kit was optimized for tissue as opposed to purified DNA further
optimization within ProMU could lead to lower CT values.
= The DEM kit demonstrates a higher p-Actin value
R Actin GSTP1
One Way ANOVA P= 0.000 One Way ANOVA P= 0.663
DEM (Mean CT) 34.990 DEM (Mean CT) 31.30
DEM STDEV 1.341 DEM STDEV 0.839
Zymo (Mean CT) 33.270 Zymo (Mean CT) 31.140
Zymo STDEV 1.240 Zymo STDEV 0.773
Number Kit (3-Actin GSTP1 APC
1 DEM 33.8 30.4 34.0
.2 DEM 34.8 30.5 33.2
3 DEM 37.8 31.8 34.1
4 DEM 35.1 32.2 33.9
5 DEM 35.7 32.5 34.4
6 DEM 35.6 32.2 34.3
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7 DEM 33.4 30.5 32.4
8 DEM 33.4 30.4 32.3
9 DEM 35.8 31.5 33.3
10 DEM 34.5 31.0 32.4
1 Zymo 32.7 32.4 33.4
2 Zymo 32.3 30.8 31.5
3 Zymo 35.6 31.9 33.3
4 Zymo 34.5 31.8 33.2
5 Zymo 34.2 31.6 32.6
6 Zymo 34.2 31.4 32.5
7 Zymo 32.1 30.4 31.5
8 Zymo 32.2 30.5 31.5
9 Zymo 32.3 30.2 31.6.
10 Zymo 32.6 30.4 31.6
The binding of crude lysate containing DNA or purified genomic DNA is
uniquely bound to the silica-gel based column utilizing the high concentration
of
salt that is present from the bisulfite conversion.
Ethanol is added to the sample prior to binding only to dissolve the
5 conversion reagent.
= Add 1/10 volume M-Dilution Buffer and incubate 70 C for 20 minutes to
chemically denature the double stranded DNA
= Add 2 volurnes of CT conversion reagent (Zymo), incubate at 70 C for 3
hours in the dark.
10 = Add 0.5 volumes of ethanol (100%) and gently vortex or pipette up and
down.
Microfuge tube containing purified DNA from urine at a starting volume of 45
NI
= Proceed with M-Dilution Buffer addition described below
= Or a microfuge tube containing (up to) 5 10 micron FFPE block slices of
a formalin fixed paraffin embedded cell culture pellet which is known to
express GSTP1 hyper Methylation.
The FFPE slices require deparaffination:
. = Add 1 ml of Xylene to the tube and mix by inverting, several times
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= Incubate at RT for 5 min and centrifuge at 13,200rpm for 5 min at RT
= Remove the supernatant without removing any pellet
= Repeat the 1 ml Xylene extraction -
= Add 1 ml EtOH (100%) to the pellet and gently mix by inverting
= Centrifuge at 13,200 rpm for 5 min at RT
= Carefully remove the EtOH by pipetting without removing any of the
pellet -
= Repeat the EtOH wash
= Dry the pellet and remove the residual ethanol in a 37 C heat block with
the microfuge tubes open for approximately 10 minutes
Extraction/modification procedure for FFPE samples
= Add 35 NI buffer ATL (Qiagen) and 10 NI of proteinase K (Qiagen) and
incubate overnight at 56 C
= After incubation the extract should be clear and homogeneous
= Proceed with M-Dilution Buffer (Zymo Research) addition described
below
= Add the sample to a Qiagen DNA purification column (QiaAmp Micro
DNA purification kit), spin 1 min at 13,200 rpm and empty waste tube;
= Add 500 NI AW1 wash buffer, spin 1 min at 13,200 rpm and empty waste
tube;
= Add 200 pl desulfonation buffer (300 mM NaOH, 90% Ethanol), incubate
20 min room temperature, spin 1 min at 13,200 rpm and empty waste
tube;
= Add 500 NI AW2 wash buffer, spin 1 min (13,200 rpm), empty waste tube
and spin 3 min (13,200 rpm).
= Elute with 20-25 pi buffer AE, TE, or Nuclease Free Water. Incubate
column for three min at room temperature and spin 9 min into new 1.5
ml microfuge tube and store at -20 C or -30 C foe =
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Example 3
Large Scale DNA Modification,
Large scale DNA modification may be necessary to make paneis for
quality control testing of methylation specific PCR methods and kits.
1. Denature 20 }ig of Prostate Cell Culture Cell line 22Rv1 genomic
DNA (ATCC) in a total volume of 225 NI TE, plus 27.5 pl of a 3.0 M NaOH
solution. Incubate 10 minutes 37 C.
2. Add 2x volume Conversion reagent (Zymo); incubate 3 hr 70 C
followed by 10 min on ice.
Bind the DNA to a solid phase support by adding 2 ml of binding buffer
containing the support matrix (Promega) and adding it to the syringe column
vacuum apparatus. Using the vacuum, filter the matrix.
Using the vacuum, wash with the matrix with 1 ml 80% IPA
Add 2 ml OD desulfonation buffer (0.3 M NaOH in 80% IPA) and
incubate at room temperature for 10 minutes. Using the vacuum, remove the
buffer.
Using the vacuum, wash with I ml 80% IPA.
3. Remove the column from the syringe and place it in a 1.5 ml
microfuge tube Elute 5 x 200 pl followed by addition of 1 ml EB
Table 5 depicts the results obtained.
Table 5
Marker Average Ct SD
Actin 33.1 0.2
GSTPI 0
APC 31.1 0.2
In this cell line the GSTPI promoter is known to be unmethylated
therefore the lack of Ct values for this marker is expected.
Example 4
A modified protocol for fast and efficient bisulfite modification of genomic
DNA
The EZ DNA Methylation Kit is provided by Zymo Research (Orange,
CA) to perform bisulfite modification of DNA, As per manufacturer's
recommendation the DNA sample to be modified is incubated with the bisulfite
conversion reagent at 50 C for 12 - 16 hrs. These conditions have been
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modified to generate comparable quality bisulfite converted DNA in much less
time. Several temperatures for different times we~e tested and demonstrated
that incubation of DNA sample with bisulfite conversion reagent at 70 C for 1-
3 hr provides efficient bisulfite modification comparable to modification
conditions recommended in the kit. The data below show methylation specific
PCR analysis with DNA samples incubated with bisulfite reagent at different
temperatures for different times.
Figure 1 shows that Veridex modified protocol of conversion at 70 C, 2
or 3hr is equivalent to manufacturer recommended 50 C for 16 hrs. This
innovation makes this procedure much faster.
Example 5
A quick and efficient protocol for DNA extraction and modification from
paraffin
Embedded Tissue
Extraction of genomic DNA and its bisulfite modification prior to being
used in a MSP reaction comprise very significant upstream procedures that are
part of this in vitro diagnostic assay. These procedures can be time consuming
involving many tedious steps and could also increase chances of sample
contamination. By combining the use of a lysis buffer, 10 mM Tris pH 8.0, 150
mM NaCI, 2 mM EDTA, 0.5% SDS including proteinase K and a bisulfite
modification kit, a quick and simple sample processing protocol to recover and
modify minimal amounts of DNA available from these sample types has been
developed.
The following steps were performed:
1. FFPE tissue (Biopsies) (5 x 10 p sections) are placed in Eppendorf
tubes.
2. Spin the tube briefly and Add 500 pi Xylene, vortex briefly at medium
speed.
3. Incubate at RT for 10 min. (During incubation at least at 2 intervals mix
the sample by inverting several times).
4. Centrifuge at full speed for 10 min at room temperature.
5. Remove supernatant by carefully decanting the liquid without losing the
pellet.
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6. Add 500 pl ethanol (100%, 200 proof) to the pellet to remove residual
Xylene. Vortex briefly at medium speed and let the tubes stand for 5 min,
mix by inverting several times.
7. Centrifuge at full speed for 10 min at room temperature.
8. Remove the ethanol by carefully decanting the liquid without losing the
pellet.
9. Repeat steps 7-10. Make sure to decant most of the liquid in this step.
10. Incubate the open microcentrifuge tube at 50 - 55 C for 10 - 15 min in
an oven to evaporate residual ethanol. Before moving to next step make
sure all ethanol has evaporated. If not incubate longer.
11. Add 40 pl of TNES lysis buffer (10 mM Tris pH 8.0, 150 mM NaCI, 2
mM EDTA, 0.5% SDS) and suspend the tissue by flicking the tube.
12. Add 10 Nl Proteinase K (20 mg/ml), vortex briefly, spin very briefly.
13. Incubate the sample at 56 C O/N in a heat block with shaking at 500
rpm.
14. Next morning spin the tubes briefly and check the tissue for complete
digestion. If there is any left over tissue add 2pI fresh Proteinase K (20
mg/ml), mix by gentle vortexing and incubate another 1 hr at 56 C, 500 rpm
in heat block.
15. Incubate tubes at 70 C x 10 min on the heat block, spin and store at -
20 C for long-term storage or proceed directly for Bisulfite modification of
extracted DNA using a commercially available DNA modification kit from
ZymoResearch.
16. Add 5 pl of M-Dilution Buffer directly to 45 pl of tissue lysate
17. Mix sample by flicking or pipetting up and down. Spin the sample
briefly. Incubate the sample at 37 C for 15 minutes in a heat block with
shaking at 1100 rpm.
During the 15 min incubation, prepare CT Conversion Reagent (as per
manufacturer's instructions).
18. After the above 15 minutes incubation, add 100 pi of the prepared CT
Conversion Reagent (after briefly spinning) to each sample and vortex
lightly (The sample may turn cloudy). Spin the sample briefly. Incubate the
sample at 70 C for 3 hr with the heating block (shaking at 1100 rpm)
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covered with aluminum foil. (The CT Conversion Reagent is light sensitive,
so try to minimize reaction's exposure to light).
19. Spin the sample down briefly. Incubate the sample on ice for 10 min.
20. Add 400 pl of M-Binding buffer to the sample and mix by pipetting up
5 and down
21. Load all the supernatant (including any precipitate) onto a Zymo-Spin I
Column and place column into a 2 ml collection tube and centrifuge at
maximum speed for 15 - 30 seconds. Discard the flow-through
22. Add 200 pl of M-Wash Buffer to the column.
10 23. Centrifuge at maximum speed for 15 - 30 second.s. Discard the
flow-through.
24. Add 200 NI of M-Desulfonation Buffer to the column and let the column
stand at room temperature for 15 minutes.
25. Centrifuge at maxirrium speed for 15 -30 seconds. Discard the*
15 flow-through.
26. Add 200 pl of M-Wash Buffer to the column.
27. Centrifuge at maximum speed for 15 - 30 seconds.
28. Add another 200 pl of M-Wash Buffer to the column.
29. Centrifuge at maximum speed for 30 sec (A longer spinning duration for
this last wash is necessary for complete removal of wash buffer residues).
Discard the flow-through.
30. Place the column into a clean 1.5 ml tube.
31. Add 25 pl of M-elution buffer directly to the column matrix. Let the
column stand for 1 min at RT. Centrifuge at maximum speed for 1 minute to
elute the DNA.
32. Store the eluted DNA at -80 C.
33. Use 5 uI in MSP reaction.
The protocol desctibed above excludes the use of a DNA purification kit
prior to bisulfite modification, thereby, reducing sample processing times,
preventing DNA losses during purification, reducing cost, and reducing chances
of contamination.
Table 6 shows that using the tissue lysate directly for bisulfite
modification of DNA gives comparable and even lower Cts than purified DNA
CA 02656327 2008-12-24
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16
using Qiagen DNA isolation kit. Thus further purification of DNA is not
required
prior to DNA modification using ZymoResearch EZ DNA methylation kit. Also,
the data show that combining TNES/PK digestion and EZ DNA methylation kit
yields more DNA sample.
Table 6
FFPE Tissue DNA extraction procedure 3-actin Ct GSTP1 Ct
2 x 5 sections Qiagen DNA isolation kit 30.1 30.9
2 x 5 11 sections TNES/PK digestion 25.4 26.7
Table 7shows that direct lysate from FFPE biopsy tissue can be used
successfully for downstream DNA modification with comparable and even
better results as compared to using Qiagen DNA isolation kit, thus avoiding
unnecessary DNA purification steps and losses.
Table 7
actin Av Cts 3 actin Av Cts
Prostate core Biopsies TNES/PK protocol Qia en DNA isolation
Sample prep 40 micron (50 micron)
J r.~ai ..`.u"e!'' ;~
~~ Z ..-110i eIUtIOf1.~N0 ~ ~ 1'
~2 YsY~,it=~ ~ FRis, 4 .s r .w.~. ,... S ZiS:'. f}r 1 R t{ "u,'
~F a ~. I.n ut,an P.C.,R~n~~ ~ ~ 5 I T~~ ~~i~~
;~
60A 34.45 33.95
6081 32.75 34.30
60B2 32.90 33.25
64A 33.30 34.20
64B 35.70 undetermined
DNA methylation assay is developed to be used on patient samples
such as archived formalin-fixed, paraffin-embedded tissues, freshly collected
urine and blood samples that comprise an invaluable resource for translational
studies of cancer and a variety of other diseases. Sample processing is a key
upstream part of this diagnostic assay. Conventionally, DNA is purified from
these sample types by using standard phenol-chloroform extraction or column
based procedures and then subjected to bisulfite modification procedures.
Several commercial kits exist for purification of DNA from paraffin embedded
archived tissues, and body fluids. However, loses during such extensive
purification procedures can significantly reduce DNA yields when very small
amount of starting tissue or body fluid sample type is available. Low DNA
yields can severely impact the downstream assay performance and can also be
time consuming. To avoid DNA loses some studies have used digested tissue
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17
lysate directly for bisulfite modification of genomic DNA using standard in
solution bisulfite modification protocols. The protocol developed in this
invention (referred to as TNES protocol) quickly and efficiently extracts and
bisulfite modifies genomic DNA by using the deproteinized lysed tissue extract
or lysed cells from urine sediment and directly using this with a
commercially.
available DNA methylation kit such as ZymoResearch EZ for downstream
bisulfite modification without any further purification steps. In addition,
the.
present invention improves multiplex PCR assay performance by minimizing
loss-during additional purification steps.
Example 6
Processing DNA samples from.urine
A. Protocol for DNA extraction from urine Samples (TNES protocol)
1. After collection, urine is to be maintained at 4 C until processed.
2. Urine is placed into a labeled 50 ml Falcon polypropylene tube and LNCap
cells (10,000 cells/50 ml) representing shedded tumor cells in a prostate
cancer patient are spiked per tube. Then the cells and particulates are
pelleted by centrifugation at 3,000 x g at 4 C.
3. Following centrifugation, carefully decant the urine supernatant into
sterile
labeled 50 ml tube and perform pellet washings in 2 steps.
4. For the first wash, the pellet is washed at 4 C with 40 ml of cold PBS in
the original 50 mi tube, gently invert the tube several times to resuspend the
pellet, and centrifuge at 3,000 x g. Aspirate the PBS wash.using a vacuum
attached to a long narrow glass or plastic tube (drawn-out Pasteur pipette or
long plastic tip) to remove as much of the wash as possible and to prevent
dislodging of the pellet over a large area of the tube (discard wash).
5. For the second wash, the pellet is resuspended in a smaller volume of 1
ml of cold PBS by gently pipetting up and down with a Pipetman. Once the
pellet is suspended, then transfer from the 50 mi tube to a 1.5 ml
microcentrifuge tube. With an additional 0.4 ml PBS, rinse the tip and the 50
ml tube to recover as much of pellet as possible and combine with the original
1 ml in the 1.5 ml tube.
6. Centrifuge at 10,000 x g for 5 min and remove the wash by vacuum
aspiration with a drawn out pipette/plastic tip. Use slight tilting of the
tube to
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18
remove as much of the liquid as possible (discard wash). The tube containing
the washed urine pellet is then placed into the sarne box as the 50 ml tube of
ciarified urine and stored at -20 C until shipping.
7. To -100 pi of washed pellet add 100 pi TNES lysis buffer (10 mM Tris pH
8.0, 150 mM NaCI, 2 mM EDTA, 0.5% SDS), incubated ~@ 56 C for 30 min.
and then stored at -20 C until processing with bisulfite modification kit (see
below, part II). In case of cells only, 20 pi TNES buffer was added to 20 NI
cell suspension.
II. Sodium Bisulfite Modification of genomic DNA using EZ-DNA methylation kit
from ZymoResearch
The EZ DNA Methylation Kit is provided by Zymo Research (Orange,
CA) to perform bisulfite modification- of DNA. As per manufacturer's
recommendation the DNA sample to be modified is incubated with the bisulfite
conversion reagent at 50 C for 12 - 16 hrs. These conditions have now been
modified to generate comparable quality bisulfite converted DNA in much less
time. Several temperatures and' different times were tested and it was
demonstrated that incubation of DNA sample with bisulfite conversion reagent
at 70 C for 1 - 3 hr provides efficient bisulfite modification comparable to
modification conditions recommended in the kit
The protocol is as follows for processing urine samples:
M-Wash Buffer (Prepare before starting using the kit)
Preparation of M-Wash buffer: Add 24 ml absolute ethanol to the M-Wash
buffer Concentrate to make the final M-Wash buffer for D5001 (Use 96 ml
Ethanol for D5002):
1. DNA modification procedure
a. Add 5 pi of M-Dilution Buffer directly to 45 pl of urine lysate or for the
higher sample volume scale up M-Dilution Buffer and urine crude lysate
proportionally. For instance, for 150 pi urine lysate add 20 pi M-dilution
buffer and 30 pi water (total of 200 NI).
b. Mix sample by flicking or pipetting up and down. Spin the sample briefly.
Incubate the sample at 37 C for 15 min in a heat block with shaking at 1100
rpm.
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19
During the 15 min incubation, prepare CT Conversion Reagent (as
per manufacturer's instructions).
c. After the above 15 minutes incubation, add 100 pl of the prepared CT '
Conversion Reagent (after briefly spinning)-to each sample (or add 400 pl
CT reagent for a scaled up protocol) and vortex lightly (the sample may turn
cloudy). Spin the sample briefly. Incubate the sample at 70 C for 3 hr with
the heating block (shaking at 1100 rpm) covered with aluminum foil. (The
CT Conversion Reagent is light sensitive, so try to minimize reaction's
exposure to light).
2. Desalting
a. Spin the sample down briefly. Incubate the sample on ice for 10 min. In
the case of the higher volume of urine sample, aliquot into two tubes of 300
NI each. *
b. Add 400 pl of M-Binding buffer to the sample and mix by pipetting up and
down or for scaled up urine samples add 800 pl of M-Binding buffer to each
aliquot, mixed and quickly spun.
c. Load all the supernatant (including any precipitate) onto a 2:ymo-Spin I
Column and place column into a 2 ml collection tube and centrifuge at
maximum speed for 15 - 30 seconds. Discard the flow-through. For a
higher sample volume, this step is repeated by adding supernatant from
each aliquoted tube one at a time on the column and centrifuging until all of
the sample is loaded onto the column.
d. Add 200 pl of M-Wash Buffer to the column.
e. Centrifuge at maximum speed for 15 - 30 seconds. Discard the
flow-through.
3. Desulfonation, 2"d desalting and elution
a. Add 200 NI of M-Desulfonation Buffer to the column and let the column
stand at room temperature for 15 minutes.
b. Centrifuge at maximum speed for 15 - 30 seconds. Discard the
flow-through.
c. Add 200 pl of M-Wash Buffer to the column.
d. Centrifuge at maximum speed for 15 - 30 seconds.
e. Add another 200 pl of M-Wash Buffer to the column.
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f. Centrifuge at maximum speed for 30 sec (a longer spinning duration for
this last wash is necessary for complete remoVal of wash buffer residues).
Discard the flow-through.
g. Place the column into a clean 1.5 ml tube.
5 h. Add 25 pl of M-elution buffer directly to the column matrix. Let the
column stand for 1 min at RT. Centrifuge at maximum speed for 1 minute to
elute the DNA..
I. Store the eluted DNA at -80 C.
j. Use 5 NI in MSP reaction.
10 Results:
The protocol described above excludes the use of a DNA purification kit
prior to bisulfite modification, thereby, reducing sample processing times,
preventing DNA losses during purification, reducing cost,'and reducing chances
=
of contamination).
15 Table 8 shows end results from MSP assay on Cepheid Smart Cycler
with DNA samples processed by TNES protocol from 50 ml of urine. Better
f3-actin and GSTP1 Cts (up to 3 Cts lower) are observed using above described
TNES/PK digestion protocol over use of purified DNA using commercially
available Qiagen DNA isolation kit (QiAmp Viral RNA kit). Thus further
20 purification of DNA is not required prior to DNA modification using this
method.
Also, the data show that combining TNES/PK digestion and EZ DNA
methylation kit yields more DNA sample.
Table 8. TNES protocol vs. Qiagen DNA isolation kit (10,000 LNCaP cells/50
ml urine)
INPUT v ac#~n Ct"vsGSTP1 Ct
extraction rotocol
50 ml Urine / 10 LNCaP cells NES 9.9 32.9
50 mi Urine / 10 LNCaP cells Qiagen 30.9 34.6.
~ ,~v rY ' u :ar. w
1;0 sLNCaP~,~scells ~ ~ NES~ W~ 21~5~~
'=~''`~'~k a "~'"~~*'' S `~39~~ i. '~~ , "'4
I LNCaP~cells
~
lfx. r=J:,~+~~ '.;~~~~F.~'.`x~i~.~.'~t:.',r~õ~ .f4i. .t~'~'4ii~
~`i`.~.c~l.,~"f~.'~~ - ~~y.,~ :'i?. L`s.l.`
Results with TNES extraction protocol are even more compelling on
serial dilutions of LNCaP prostate cells (range of 10,000 to 100 spiked per 50
ml pooled urine from healthy donors). Combined protocol for DNA extraction
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21
followed directly by bisulfite modification allows to improve sensitivity of
the
prostate methylation assay by 10 fold (Table 9) as compared with two
commercial kits combined. TNES protocol allows detection of 100 cells per
50 ml urine, the level which is undetectable by Qiagen protocol using both Ct
value and copy number analysis.
Table 9. TNES protocol vs. Qiagen DNA isolation kit (LNCaP cells: 10,000,
1000 and 100/50 ml'urine).
TNES-ZR BMK protocol Qia en-ZR BMK protocol
Task GSTP GSTP Ratio Gst-Pi GST GSTP1 Ratio Gst-Pi
# cells/mi Ct Copies M/Q-actin P1 Ct Copies M/G3-actin
urine co ies X 1000 co ies X 1000
/50 29.70 6365 487 30.88 3051 575
10 /50 31.10 2651 113 33.63 546 68
10 /50 31.38 2232 126 33.83 482 58
10/50 35.05 224 17 38.93 20 2
10 /50 .34.43 331 22 38.58 25 3
10I50 38.60 24 2 00.00 0 0
10 /50 0.00 0 0 00.00 0 0
Ct values, Copy numbers and methylation ratios are presented for sample
replicates of the same cell load in 50 mi urine
Although the foregoing invention has been described in some detail by
10 way of illustration and example for purposes of clarity of understanding,
the
descriptions and examples should not be construed as limiting the scope of the
invention.
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22
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