S7804_技术文档

CpG WIZ™ p16 Amplification Kit

S7800

CpG WIZ™ p15 Amplification Kit

S7802

CpG WIZ™ E-cadherin

Amplification Kit

S7804

CpG WIZ™ Prader-Willi/Angelman

Amplification Kit

S7806

FOR RESEARCH USE ONLY

Not for use in diagnostic procedures

USA & Canada

Phone: +1(800) 437-7500 • Fax: +1 (909) 676-9209 • Europe +44 (0) 23 8026 2233

Australia +61 3 9839 2000 • Germany +49-6192-207300 • ISO Registered Worldwide

www.chemicon.com • custserv@chemicon.com • techserv@chemicon.com

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TABLE OF CONTENTS

I. INTRODUCTION ..............................................................................1

Using this Manual..................................................................................... 1

Background............................................................................................... 1

Principles of the Technique....................................................................... 2

Fig 1: DNA Treatment with Sodium Bisulfite....................................... 3

II. KIT COMPONENTS .........................................................................4

Materials Required But Not Supplied ....................................................... 5

III. PROTOCOLS....................................................................................7

CpG WIZ™ p16 (S7800), p15 (S7802) and

E-cadherin (S7804) Amplification Kits ................................................. 7

Experimental Design............................................................................ 7

Fig. 2: Specificity of the CpG WIZ™ p16

Amplification Kit.................................................................................. 9

Amplification Protocol....................................................................... 10

CpG WIZ™ Prader-Willi/Angelman (S7806) Amplification Kit........... 12

Experimental Design.......................................................................... 12

Fig. 3: Specificity of the CpG WIZ™ Prader-Willi/

Angelman Amplification Kit............................................................... 14

Amplification Protocol....................................................................... 15

IV. DATA ANALYSIS ...........................................................................18

CpG WIZ™ p16 (S7800), p15 (S7802) and

E-cadherin (S7804) Amplification Kits ............................................. 18

CpG WIZ™ Prader-Willi/Angelman (S7806) Amplification Kit........... 19

V. TROUBLESHOOTING ....................................................................20

CpG WIZ™ p16 (S7800), p15 (S7802) and

E-cadherin (S7804) Amplification Kits .................................................. 20

CpG WIZ™ Prader-Willi/Angelman (S7806) Amplification Kit........... 21

i

VI. APPENDIX ............................................................................ 23

Laboratory Setup and Precautions .......................................................... 23

Related Products...................................................................................... 23

VII. REFERENCES ................................................................................24

References Cited in this Manual ............................................................. 24

Disclaimers ............................................................................................ 25

Warranty.................................................................................................. 25

ii

I. INTRODUCTION

Using This Manual

Please read the entire instruction manual prior to using CpG WIZ™

Amplification Kits. Note that in the Procedures and Troubleshooting sections,

instructions are separate for the CpG WIZ™ Prader-Willi/ Angelman

Amplification Kit (S7806). Should additional questions arise, assistance is

available from Chemicon Technical Service at techserv@chemicon.com or

(800) 437-7500.

Background

Methylation of cytosines located 5' to guanosine is known to have a profound

effect on the expression of several eukaryotic genes (1). In normal cells,

methylation occurs predominantly in CG-poor regions, while CG-rich areas,

called CpG islands, remain unmethylated. The exception is extensive

methylation of CpG islands associated with transcriptional inactivation of

regulatory regions of imprinted genes (2, 3) such as those associated with

Prader-Willi/Angelman Syndrome (4) and genes on the inactive X-chromosome

of females (5, 6). Aberrant methylation of normally unmethylated CpG islands

has been documented as a relatively frequent event in immortalized and

transformed cells (7) and has been associated with transcriptional inactivation of

defined tumor suppressor genes in human cancers (8, 9). E-cadherin, p16, and

p15 are examples of genes that exhibit characteristic hypermethylation.

Previously developed methods to determine the methylation status of cytosine

include digestion with methylation sensitive restriction enzymes and genomic

DNA sequencing. Both techniques have limitations: restriction enzymes can

only detect methylation sites within their recognition sequence and sequencing

is time consuming. Increasing the detection sensitivity of CpG island

methylation has the potential to define tumor suppressor gene function and

provides a new strategy for early tumor detection.

Methylation-specific PCR (MSP) is a new technology for sensitive detection of

abnormal gene methylation utilizing small amounts of DNA (10). This process

employs an initial bisulfite reaction to modify the DNA, followed by PCR

amplification with specific primers designed to distinguish methylated from

unmethylated DNA. The CpGenome™ DNA Modification Kit (S7820) contains

the reagents necessary to perform the initial bisulfite reactions, while CpG

WIZ™ Amplification Kits contain the reagents required for the PCR

amplification reactions.

1

Principles of the Technique

MSP, performed using the CpGenome™ DNA Modification Kit and CpG

WIZ™ Amplification Kits, permits sensitive detection of altered DNA. Due to

the fact that it is a PCR-based assay, it is extremely sensitive, facilitating the

detection of low numbers of methylated alleles and the study of samples

containing low amounts of DNA. MSP also allows examination of all CpG sites,

not just those within sequences recognized by methylation sensitive restriction

enzymes. Increasing the number of such sites which can be assessed allows

rapid, fine mapping of methylation patterns throughout CpG regions. In

addition, the bisulfite modification is ideally suited for analysis of CpG islands

since it converts the majority of cytosines to uracils, making a region of the

genome which is CG-rich less difficult to amplify by PCR.

Methylation-specific PCR employs an initial bisulfite reaction to modify the

DNA, followed by a "hot start" PCR amplification with specific primers

designed to distinguish methylated DNA from unmethylated DNA. As shown in

Figure 1, in the bisulfite reaction, all unmethylated cytosines are converted to

uracils while 5-methylcytosines remain unaltered. Thus, the sequence of the

treated DNA will differ if the DNA is originally methylated vs. unmethylated.

Primers contained in CpG WIZ™Amplification Kits are designed to specifically

amplify each of the sequences based upon these chemically-induced differences.

If the sample DNA was originally unmethylated, a product will be generated

after PCR using the U primer set. Conversely, a product will be generated using

the M primer set if the sample was originally methylated.

2

Figure 1: DNA Treatment with Sodium Bisulfite.

Unmethylated DNA

Methylated DNA

3

II. KIT COMPONENTS

The components of CpG WIZ™ Amplification Kits include those required for

PCR amplification after bisulfite modification of DNA samples. Sufficient

reagents are provided to analyze 25 samples with appropriate controls.

Table 1: CpG WIZ™ p16 (S7800), p15 (S7802) and E-cadherin (S7804)

Amplification Kit Components (color-coded microcentrifuge tube caps)

Storage

Description

Amount

Conditions

U Primer Set

35 µL (white cap)

35 µL (red cap)

-15°C to -25°C

5 µM each primer (25X)

M Primer Set

-15°C to -25°C

5 µM each primer (25X)

W Primer Set

35 µL (green cap)

50 µL (white cap)

50 µL (red cap)

5 µM each primer (25X)

U control DNA

0.1 µgµL

M control DNA

0.1 µg/µL

W control DNA

0.05 µg/µL

50 µL (green cap)

265 µL (blue cap)

-15°C to -25°C

Universal 10X PCR Buffer

4

Table 2: CpG WIZ™ Prader-Willi/Angelman (S7806) Amplification Kit

Components (color-coded microcentrifuge tubes)

Storage

Description

Amount

Conditions

P (paternal) primer set

5 µM each primer (25X)

M (maternal) primer set

5 µM each primer (25X)

Normal control DNA

0.1 µg/µL

70 µL (white cap)

70 µL (red cap)

20 µL (green cap)

175 µL (blue cap)

-15°C to -25°C

10X PCR buffer

Materials Required But Not Supplied

Equipment and Supplies

a. Thermocycler

b. Gel electrophoresis apparatus (vertical or horizontal)

c. Power Supply

d. Screw-cap tubes for PCR amplification

e. Aerosol-resistant pipette tips

f. Microcentrifuge (to 12,000 X g)

g. 302 nm UV transilluminator, camera and film

Reagents

a 2.5 mM dNTP mix (2.5 mM of each nucleotide)

b. Taq polymerase

Note: For S7806 (CpG WIZTM Prader-Willi/Angelman Amplification Kit),

the use of a "hot start" enzyme is strongly recommended. See Sec. III.

Protocols.

c. "Hot start" PCR reagents for S7800, S7802, and S7804 (see Sec. III.

Protocols).

5

d. Reagents for gel electrophoresis (2% agarose, 10% acrylamide, or suitable

high resolution agarose)

e. DNA markers (size range 100-300 bp)

f. Ethidium bromide (10 mg/mL)

g. Gel-loading solution

h. Bisulfite Modified DNA (CpGenome™ DNA Modification Kit, S7820)

6

III. PROTOCOLS

CpG WIZ™ p16 (S7800), p15 (S7802) and E-cadherin (S7804)

Amplification Kits

Experimental Design

Primer Sets

CpG WIZ™ Amplification Kits contain primers that can be used for analysis of

DNA samples by MSP. However, the samples must first undergo bisulfite

modification prior to PCR amplification. CpGenome™ DNA Modification Kit,

S7820, contains the reagents necessary to perform the modification. Chemical

modification creates the sequence differences between the methylated and

unmethylated DNA. The primer sets in the kit are engineered to anneal to the

DNA, based upon the sequence differences.

U Primer Set will anneal to unmethylated DNA that has undergone a chemical

modification

M Primer Set will anneal to methylated DNA that has undergone a chemical

modification

W Primer Set serves as a control for the efficiency of chemical modification. It

will anneal to any DNA (unmethylated or methylated) that has NOT undergone

chemical modification, hence, the "wild type", or W.

Data interpretation can still proceed in the case of incomplete chemical

modification (up to 50%).

Amplification Regions

The amplified region is defined as the sequence between the 3' nucleotide of the

sense primer and the complement of the 3' nucleotide of the anti-sense primer

for each gene promoter. The nucleotide numbering systems are those used in the

GenBank submissions identified by the following accession numbers: p16,

X94154; p15, S75756; E-cadherin, L34545.

7

“Hot Start” PCR

The three sets of primers used in the CpG WIZ™ Methylation Assay are derived

from sequences closely related to each other, which introduces the possibility of

mispriming. In order to avoid this and other PCR-related artifacts, "hot start"

PCR is recommended. "Hot start" PCR permits the Taq polymerase to begin the

reaction only after the template and primers are in single-stranded form.

There are several modifications of the standard PCR protocol which allow a "hot

start" to occur. In one scenario, the PCR reaction mixture excluding the

polymerase can be overlaid with mineral oil prior to heating to 95°C. At the end

of the incubation, the enzyme is pipetted directly into the mixture under the

mineral oil. A second method involves the physical separation of the polymerase

and the rest of the PCR mix with a wax bead. The enzyme combines with the

rest of the reaction mixture only after the wax melts. In another variation, an

anti-Taq antibody inhibits the polymerase during reaction setup by forming a

complex with the Taq enzyme. Taq polymerase becomes active when the

complex is abolished due to antibody denaturation during the 95°C incubation.

Alternatively, a "hot start" enzyme can be used. Refer to the manufacturer's

instructions for enzyme activation protocol.

Note: Do not use a polymerase with 3'-5' exonuclease activity (i.e.

proofreading).Do not use a wax bead that contains Mg²⁺. Any extra Mg²⁺added

to the reaction mixture produces suboptimal results.

Genomic Control DNAs

The methylated (red cap) and unmethylated (white cap) control DNAs must

undergo bisulfite modification prior to PCR amplification using the

CpGenome™ DNA Modification Kit (S7820). When used with their respective

U and M primer sets, a single PCR product of an expected size is obtained in

each case.

W control genomic DNA is used in the PCR and is NOT to be used in the

chemical modification step. When used with the W primer set, it is a positive

control for PCR amplification. Failure to generate a PCR product indicates a

general failure in the PCR reaction.

8

Specificity of the Assay

The specificity of the CpG WIZ™ p16 Amplification Kit is shown in Figure 2.

With a complete chemical modification reaction, U primers amplify only

unmethylated DNA (154 bp, lane 1) and M primers amplify only methylated

DNA (145 bp, lane 5). W primers amplify only DNA which is not chemically

modified, or "wild type W" (142 bp, lane 9).

Figure 2: Specificity of the CpG WIZ™ p16 Amplification Kit.

M 1 2 3 4 5 6 7 8 9 10 11 12 M

Polyacrylamide gel analysis using the primers and the control DNA samples

included in the kit is shown. (Lanes 1-3: U control DNA; lanes 4-6: M control

DNA; lanes 7-9: W control DNA; lanes 10-12: Minus DNA control. Lanes 1, 4,

7, 10: U primers; lanes 2, 5, 8, 11: M primers; lanes 3, 6, 9, 12: W primers. M

lane: 100 base-pair marker.)

Experiment Setup

CpG WIZ™ Amplification Kits (S7800, S7802, and S7804) include sufficient

reagents to analyze 25 samples with appropriate controls (105 PCR reactions).

Each experiment will include chemical modification of seven DNAs: 5

experimental DNA and 2 control DNA samples, (M and U). In the subsequent

PCR reactions, each chemically modified experimental DNA sample is

amplified with each of three oligonucleotide primer sets U, M and W. The

chemically modified genomic control DNAs, U and M, are amplified with their

corresponding primer set. Untreated W genomic control DNA is amplified with

the W primer set. Lastly, a negative PCR control (i.e. no DNA) is performed for

each set of primers.

9

For example, a typical gel for the analysis of five experimental DNA samples

includes a total of 21 lanes:

Lanes 1-3

Lanes 4-6

Lanes 7-9

Lanes 10-12

Lanes 13-15

Lane 16

Experimental sample 1 with U, M and W primers

Experimental sample 2 with U, M and W primers

Experimental sample 3 with U, M and W primers

Experimental sample 4 with U, M and W primers

Experimental sample 5 with U, M and W primers

Chemically modified control U DNA with U primers

Chemically modified control M DNA with M primers

Untreated control W DNA with W primers

Lane 17

Lane 18

Lanes 19-21

No DNA control with U, M, and W primers

Amplification Protocol

To prevent PCR contamination, read Sec. VI. Appendix, Laboratory Setup and

Precautions before beginning.

STEP 1. Modification

Prior to performing PCR with the primer sets provided in CpG WIZ™

Amplification Kits, one microgram of purified DNA must undergo bisulfite

modification with the reagents contained in CpGenome™ DNA Modification

Kit (S7820).

STEP 2. Amplification

"Hot start" PCR is recommended for this assay (refer to Sec. III. Protocols,

Experimental Design). This is accomplished by several mechanisms, including a

wax barrier or anti-Taq antibody. Refer to the instructions specified by the

manufacturer of the "hot start" PCR reagents, and modify the amplification

"master mix" and reaction conditions accordingly in steps b-f, below.

a. Determine the number of assays to be run in the experiment: run three

amplification reactions for each experimental DNA sample plus six control

reactions per each set of methylation assays (refer to Sec. III. Protocols,

Experimental Design).

b. Prepare three (3) "master mixes" which correspond to the 3 possible primer

sets U, M and W (primer cap colors-white, red and green) by mixing all the

reagents outlined below except for the template DNA.

10

To analyze five experimental samples with appropriate controls, use the

following amount of "master mixes" of each color: 7 tubes X 23.0 µL = 161 µL,

plus 10% of that volume to adjust for pipetting error. Multiply the volume of

each reagent listed below by 7 and add 10% for each "master mix" (white, red

and green). Thaw all reagents and store on ice while creating the "master

mixes". The amount of reagents required in each reaction is:

10X Universal PCR buffer

2.5 mM dNTP Mix**

2.5 µL

U, M or W primers (white, red and green)

TaKaRa™ Taq or "hot start" enzyme (5 U/µL)

dH₂O

1.0 µL

0.2 µL (1 Unit)

16.8 µL

23.0 µL

Template DNA (50 ng/µL)

TOTAL VOLUME

2.0 µL

25.0 µL

** Upon first use, make aliquots of 2.5 mM dNTPs, which should be freeze-

thawed no more than 5 times.

c. Aliquot 23 µL of each "master mix" (white, red and green) into

corresponding PCR tubes.

d. Add:

2 µL of water to the no DNA control tube.

2 µL of modified sample DNA to each of the sample tubes.

2 µL of corresponding DNA controls (modified U and M, unmodified W) to

each control tube.

e. Place tubes in the thermocycler block, and perform PCR under the following

conditions:

Denature:

for Taq Polymerase

95°C / 5 minutes

for "hot start" enzyme check manufacturer's specifications

Then, perform 35 cycles of the following conditions:

denature

anneal

95°C / 45 seconds

60°C / 45 seconds

72°C / 60 seconds

extend

11

f. Remove the tubes from the thermocycler block. From this point on, it is

important to designate separate pipettes and work areas for amplified vs.

unamplified samples. This prevents carry-over contamination of future DNA

samples with the amplified product.

STEP 3. Gel Electrophoresis

a. After the completion of PCR, add an appropriate amount of loading dye to

the sample and analyze 5 µL of the reaction on a 2% agarose, or a 10%

native acrylamide or other high resolution agarose gel. Use DNA markers

(100-300 bp range) to determine the size of PCR products.

b. After electrophoresis, stain the gel with ethidium bromide. Dilute the 10

mg/mL stock solution 1:10,000 in deionized water. Stain for 10-30 minutes

and destain for 10-30 minutes in deionized water at room temperature.

Note: Ethidium bromide is a known carcinogen. Exercise appropriate

caution and good lab practice when using this reagent.

CpG WIZ™ Prader-Willi/Angelman (S7806) Amplification Kit

Experimental Design

Primer Sets

The CpG WIZ™ Prader-Willi/Angelman Amplification Kit contains primers

that can be used for analysis of DNA samples by MSP. However, the first step

of MSP is the bisulfite modification of the DNA samples. CpGenome™ DNA

Modification Kit (S7820), contains the reagents necessary to perform the

modification. This chemical modification creates the sequence differences

between the methylated and unmethylated DNA. The primer sets in the kit are

engineered to anneal to the DNA, based upon the sequence differences.

P (Paternal) Primer Set will anneal to unmethylated DNA that has undergone a

chemical modification.

M (Maternal) Primer Set will anneal to methylated DNA that has undergone a

chemical modification.

12

Amplified Regions

The P and M primer sets are designed to amplify a differentially methylated site

present at the CpG island of the small nuclear ribonucleoprotein-associated

polypeptide N (SNRPN), a candidate gene for Prader-Willi Syndrome. The

amplified region is defined as the sequence between the 3' nucleotide of the

sense primer and the complement of the 3' nucleotide of the anti-sense primer

for each primer pair. The nucleotide numbering system is that used in the

GenBank submission identified by the accession number L32702.

"Hot Start" PCR

The two sets of primers used in the CpG WIZ™ Prader-Willi/Angelman

Amplification Kit are derived from sequences closely related to each other,

which introduces the possibility of mispriming. In order to avoid this and other

PCR-related artifacts, it is recommended that a "hot start" enzyme be used.

Normal Control DNA

The normal control contains DNA from both maternal and paternal homologs

which means that both methylated and unmethylated SNRPN sequences are

represented. Therefore, PCR products will be obtained after the bisulfite

modification of the normal control DNA when using either the M or the P

primer sets. The M primer set will generate a 174 bp product while the P primer

set produces a 100 bp fragment. Multiplex amplification is feasible since the

fragment sizes are sufficiently dissimilar.

Specificity of the Assay

The specificity of the CpG WIZ™ Prader-Willi/Angelman Amplification Kit is

shown in Figure 3. With a complete chemical modification reaction, P primers

amplify only unmethylated DNA (100 bp, lane 1) and M primers amplify only

methylated DNA (174 bp, lane 2). The smaller bands present in the lanes where

the M primer set was used in the PCR reaction represent excess primers.

13

Figure 3: Specificity of the CpG WIZ™ Prader-Willi/Angleman Amplification

Kit.

M

1

2

3

4

Polyacrylamide gel analysis using the primers and the control DNA samples

included in the kit is shown. (Lane 1: P primer set with modified control DNA;

Lane 2: M primer set with modified control DNA; Lane 3: P primer set with

unmodified control DNA; Lane 4: M primer set with unmodified control DNA.

Marker Lane: 100 bp ladder)

Experiment Setup

The CpG WIZ™ Prader-Willi/Angelman Amplification Kit includes sufficient

reagents to analyze 25 samples with appropriate controls (70 PCR reactions).

For each batch of experimental DNA samples to be analyzed, the experimental

samples and the normal control DNA must first undergo bisulfite modification.

In the subsequent PCR reactions, the chemically modified DNA samples, both

experimental and control, are amplified using both the maternal (M) and

paternal (P) primer sets. In addition, a negative PCR control (i.e. no DNA) is

performed for each set of primers.

For example, a typical gel for the analysis of five experimental DNA samples

and proper controls includes a total of 14 lanes:

Lanes 1-2

Lanes 3-4

Lanes 5-6

Experimental sample 1 with P and M primers

Experimental sample 2 with P and M primers

Experimental sample 3 with P and M primers

14

Lanes 7-8

Lanes 9-10

Lane 11

Experimental sample 4 with P and M primers

Experimental sample 5 with P and M primers

Chemically modified normal control DNA with P primers

Chemically modified normal control DNA with M primers

No DNA Control with P and M primers

Lane 12

Lanes 13-14

Amplification Protocol

To prevent PCR contamination, read Sec. VI. Appendix, Laboratory Setup and

Precautions before beginning.

STEP 1. Modification

Prior to performing PCR with the primer sets in the CpG WIZ™ Prader-

Willi/Angelman Amplification Kit, the DNA samples must undergo bisulfite

modification. CpGenome™ DNA Modification Kit (S7820), contains the

reagents necessary to perform the modification.

STEP 2. Amplification

a. Determine the number of assays to be run in the experiment: run two

amplification reactions for each experimental DNA sample plus four control

reactions per each set of methylation assays (see Sec. III. Protocols,

Experimental Design).

b. Prepare two (2) "master mixes" which correspond to the 2 possible primer

sets P and M (primer cap colors-white and red) by mixing all the reagents

outlined below except for the template DNA.

To analyze five experimental samples with appropriate controls, use the

following amount of "master mixes" of each color: 7 tubes X 23.0 µL = 161 µL,

plus 10% of that volume to adjust for pipetting error. Multiply the volume of

each reagent listed below by 7 and add 10% for each "master mix" (white and

red). Thaw all reagents and store on ice while creating the "master mixes".

15

The amount of reagents required in each reaction is:

10X Universal PCR buffer

2.5 mM dNTP Mix**

P or M primers (white, red)

"Hot start" enzyme (5 U/µL)

dH₂O

2.5 µL

2.0 µL

0.1 µL (0.5 U)

16.4 µL

23.0 µL

Template DNA (50 ng/µL)

TOTAL VOLUME

2.0 µL

25.0 µL

** Upon first use, make aliquots of 2.5 mM dNTPs, which should be freeze-

thawed no more than 5 times.

c. Aliquot 23 µL of each "master mix" (white and red) into corresponding PCR

tubes.

d. Add:

2 µL of water to the no DNA control tube.

2 µL of modified sample DNA to each of the sample tubes.

2 µL of modified normal DNA control to each control tube.

e. Place tubes in the thermocycler block, and perform PCR under the following

conditions:

Denature:

for "hot start" enzyme check manufacturer's specifications

Then, perform 35 cycles of the following conditions:

denature

anneal

95°C / 30 seconds

62°C / 30 seconds

72°C / 30 seconds

extend

Final Extension:

72°C /10 minutes

f. Remove the tubes from the thermocycler block. From this point on, it is

important to designate separate pipettors and work areas for amplified vs.

unamplified samples. This prevents carry-over contamination of future DNA

samples with the amplified product.

16

STEP 3. Gel Electrophoresis

a. After the completion of PCR, add an appropriate amount of loading dye to

the sample and analyze 5 µL of the reaction on a 2% agarose or a 10% native

acrylamide or other high resolution agarose gel. Use DNA markers (100-300

bp range) to determine the size of PCR products.

b. After electrophoresis, stain the gel with ethidium bromide. Dilute the 10

mg/mL stock solution 1:10,000 in deionized water. Stain for 10-30 minutes

and destain for 10-30 minutes in deionized water at room temperature.

Note: Ethidium bromide is a known carcinogen. Exercise appropriate

caution and good lab practice when using this reagent.

17

IV. DATA ANALYSIS

CpG WIZ™ p16 (S7800), p15 (S7802) and E-cadherin (S7804)

Amplification Kits

Table 3: Sizes of Expected Products from Controls

Controls

p16

154

145

142

p15

162

154

137

E-cadherin

212

U primer/U control DNA

M primer set/M control DNA

W primer set/W control DNA

206

194

In the three no DNA control lanes, no PCR products should be generated.

Table 4: Sizes of Expected Products from Samples

Experimental Samples

p16

154

145

p15

162

154

E-cadherin

212

U primer set/ unmethylated

DNA

M primer set/methylated DNA

206

If the sample is a mixture of unmethylated and methylated DNA, both the U and

M primer will produce a PCR product.

If the W primer set produces a PCR product with an experimental sample, it is

an indication of incomplete chemical modification.

18

CpG WIZ™ Prader-Willi/Angelman (S7806) Amplification Kit

Table 5: Sizes of Expected Products

Primer Set

Template

P

100

—

M

174

—

Normal Control DNA

Experimental Methylated DNA

Experimental Unmethylated DNA

100

In the two no DNA control lanes, no PCR products should be generated.

In a clinical research sample obtained from a normal individual, a 100 bp and a

174 bp fragment will be obtained when using the P primer set and M primer set,

respectively, in PCR reactions. MSP performed on a research sample from an

individual with Prader-Willi Syndrome will produce the 174 bp fragment with

the M primer set but will generate no product with the P primer set. Conversely,

a research sample from an individual with Angelman Syndrome will produce the

100 bp fragment with the P primer set, but will generate no product with the M

primer set.

19

V.

TROUBLESHOOTING

CpG WIZ™ p16 (S7800), p15 (S7802) and E-cadherin (S7804)

Amplification Kits

There is no visual evidence of products in any lane.

?

Potential Problem: PCR amplification is not initiated.

Recommendations:

a. Confirm that all PCR components were added to the reaction tube.

b. Confirm that the time and temperature settings on the thermocycler match

those described in this manual.

c. If performing "hot start" PCR using a "hot start" enzyme, verify the initial

denaturation/activation time of 12 minutes at 95°C.

d. For all other "hot start" methods, confirm the proper use of the reagents.

e. Confirm that the PCR polymerase is still active.

f. Confirm that no additional Mg²⁺was added to the PCR reaction mix.

g. The optimal annealing temperature is 60°C. If items #a-f have not remedied

the problem, re-optimize annealing conditions to suit your amplification

instrument.

No amplification product is generated in the experimental

samples using U, M and W primer sets, but products of the

?

correct size are observed with the control samples.

Potential Problem #1: Experimental DNA samples were degraded

prior to chemical modification.

Recommendation:

Purify the genomic DNA again and repeat the chemical modification.

Potential Problem #2: Chemically modified experimental DNA

samples were stored for more than two months prior to PCR.

Recommendation:

Repeat the chemical modification on new genomic DNA samples.

U or M primer sets are producing bands in all samples,

including the "no DNA" controls.

?

Potential Problem: PCR reagents are contaminated with

amplification products.

20

Recommendations: see Sec. VI. Appendix, Laboratory Setup and Precautions.

a. Use fresh aliquots of every PCR component (i.e. dNTPs, buffer, etc.)

b. Use separate sets of pipettors for pre- vs. post-amplification liquid

dispensing.

c. Devote a work area to pre- and post-amplification procedures.

d. Always use aerosol-resistant pipette tips.

e. Always use a clean labcoat and gloves.

W primer set produces an amplification product in some or

all experimental samples, in addition to an amplification

?

product from the U or M primer set.

Potential Problem: Chemical modification of the experimental DNA

sample(s) is incomplete.

Recommendation:

This will not jeopardize the validity of the assay as long as a product is also

produced using the U or M primer set. The PCR product produced with the W

primer set is always smaller than that produced with either U or M.

The only lanes containing a PCR product are from those

DNA samples (experimental and control) amplified with

?

the W primer set.

Potential Problem: Chemical modification of the experimental DNA

samples did not work.

Recommendation:

If using the CpGenome™ DNA Modification Kit (S7820), check the

troubleshooting section of the manual.

CpG WIZ™ Prader-Willi/Angelman (S7806) Amplification Kit

There is no visual evidence of products in any lane.

?

Potential Problem: PCR amplification is not initiated.

Recommendations:

a. Confirm that all PCR components were added to the reaction tube.

b. Confirm that the time and temperature settings on the thermocycler match

those described in this manual.

21

c. If performing "hot start" PCR using a "hot start" enzyme, verify the initial

denaturation/activation time of 12 minutes at 95°C.

d. Confirm that the PCR polymerase is still active.

e. Confirm that no additional Mg²⁺was added to the PCR reaction mix.

f. The optimal annealing temperature is 62°C. If items #a-e have not remedied

the problem, re-optimize annealing conditions to suit your amplification

instrument.

No amplification product is generated in the experimental

samples using the P and M primer sets, but products of the

?

correct size are observed with the control samples.

Potential Problem #1: Experimental DNA samples were degraded

prior to chemical modification.

Recommendation:

Purify the genomic DNA again and repeat the chemical modification.

Potential Problem #2: Chemically modified experimental DNA

samples were stored for more than two months prior to PCR.

Recommendation:

Repeat the chemical modification on new genomic DNA samples.

P or M primer sets are producing bands in all samples,

including the "no DNA" controls.

?

Potential Problem: PCR reagents are contaminated with

amplification products.

Recommendations: see Sec. VI. Appendix, Laboratory Setup and Precautions

a. Use fresh aliquots of every PCR component (i.e. dNTPs, buffer, etc.)

b. Use separate sets of pipettors for pre- vs. post-amplification liquid

dispensing.

c. Devote a work area to pre- and post-amplification procedures.

d. Always use aerosol-resistant pipette tips.

e. Always use a clean labcoat and gloves.

22

VI. APPENDIX

Laboratory Setup and Precautions

One of the most important considerations when performing MSP using CpG

WIZ™ Amplification Kits is the environment where the initial reaction mixtures

are set up. The ideal environment is free of amplified DNA products, which can

cause false-positive results. Potential sources of PCR product contamination are:

contaminated pipettors and tips, gel box and buffer, tube racks, notebooks, lab

coats and any other item exposed to amplified PCR products.

The following precautions should be followed in all steps of the assay protocol:

a. Always wear gloves.

b. Use sterile water for all solutions, aliquot the solutions in small amounts, and

use fresh aliquots as working solutions. Discard working solutions after use.

c. Keep the assay solutions (10X PCR buffers, dNTPs, polymerase, etc.)

separate from the amplified DNA.

d. Always use aerosol resistant pipette tips.

e. Separate micropipettors and work areas are recommended for the following

three steps of the assay:

1. DNA modification and purification

2. Amplification setup

3. Post-amplification analysis


型号：	S7804
厂家：	ETC
描述：	CpG WIZ Prader-Willi/Angelman Amplification Kit 的CpG WIZ普拉德威利/安琪儿扩增试剂盒
文件：	总32页 (文件大小：357K)
中文：	中文翻译
下载：	下载PDF数据表文档文件

S7804 [ETC]

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