Bio-Rad DCode Universal Mutation Detection System User Manual

The DCode

™

Universal Mutation

Detection System

Catalog Numbers

170-9080 through 170-9104

For technical service call your local Bio-Rad office or in the U.S., call 1-800-4BIORAD (1-800-424-6723)

Warranty

The DCode universal mutation detection system lid, tanks, casting stand, gradient mixer, and accessories are warranted against defects in materials and workmanship for 1 year. If any defects occur during this period, Bio-Rad will repair or replace the defective parts at our discretion, without charge. The following defects, however, are excluded:

1. Defects caused by improper operation.

2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized agent.

3. Damage caused by substituting alternative parts.

4. Use of fittings or spare parts supplied by anyone other than Bio-Rad Laboratories.

5. Damage caused by accident or misuse.

6. Damage caused by disaster.

7. Corrosion caused by improper solvent†or sample.

This warranty does not apply to parts listed below:

• Fuses

• Glass plates

• Electrodes

For any inquiry or request for repair service, contact Bio-Rad Laboratories. Inform Bio-Rad of the model and serial number of your instrument.

Important: This Bio-Rad instrument is designed and certified to meet EN61010-1* safety standards. Certified products are safe to use when operated in accordance with the instruction manual. This instrument should not be modified or altered in any way. Alteration will:

• Void the manufacturer’s warranty

• Void the EN61010-1 safety certification

• Create a potential safety hazard

Bio-Rad Laboratories is not responsible for any injury or damage caused by the use of this instrument for purposes other than those for which it is intended, or by modifications of the instrument not performed by Bio-Rad Laboratories or an authorized agent.

The Model 475 Gradient Delivery System is covered by U.S. patent number 5,540,498.

Practice of PCR is covered by U.S. patent numbers 4,683,195; 4,683,202; 4,899,818 issued to Cetus Corporation, which is a subsidiary of Hoffmann-LaRoche Molecular Systems, Inc. Purchase of any of Bio-Rad’s PCR-related products does not convey a license to use the PCR process covered by these patents. To perform PCR, the user of these products must obtain a license.

† The DCode system tank is not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene,

benzene), or acetone. Use of organic solvents voids all warranties.

* EN61010-1 is an internationally accepted electrical safety standard for laboratory instruments.

Table of Contents

Section 1 General Safety Information ..........................................................1

1.1 Caution Symbols........................................................................................1

1.2 Precautions During Set-up .........................................................................1

1.3 Precautions During a Run..........................................................................1

1.4 Precautions After a Run.............................................................................2

1.5 Safety ........................................................................................................2

Section 2 Introduction ...................................................................................2

2.1 Introduction to Mutation Detection Technology...........................................2

Section 3 Product Description......................................................................2

3.1 Packing List ...............................................................................................2

3.2 System Components and Accessories.......................................................5

Section 4 Denaturing Gel Electrophoresis (DGGE, CDGE, TTGE)............11

4.1 Introduction to Denaturing Gradient Gel Electrophoresis (DGGE) ............11

Reagent Preparation................................................................................13

Gel Volumes ............................................................................................15

Sample Preparation .................................................................................16

Temperature Controller............................................................................17

Preheating the Running Buffer.................................................................18

Assembling the Perpendicular Gradient Gel Sandwich ............................18

Casting Perpendicular Gradient Gels .......................................................22

Assembling the Parallel Gradient Gel Sandwich ......................................24

Casting Parallel Gradient Gels .................................................................26

4.2 Introduction to Constant Denaturing Gel Electrophoresis (CDGE)............28

Reagent Preparation................................................................................29

Gel Volumes ............................................................................................31

Sample Preparation .................................................................................31

Temperature Controller............................................................................32

Preheating the Running Buffer.................................................................33

Assembling the CDGE Gel Sandwich ......................................................33

Casting CDGE Gels.................................................................................35

4.3 Introduction to Temporal Temperature Gradient Gel Electrophoresis

(TTGE) ....................................................................................................36

Calculating the Run Parameters ..............................................................37

Reagent Preparation................................................................................38

Gel Volumes ............................................................................................40

Sample Preparation .................................................................................40

Preheating the Running Buffer.................................................................40

Assembling the TTGE Gel Sandwich .......................................................40

Temperature Controller............................................................................41

Casting TTGE Gels..................................................................................43

Section 5 Heteroduplex Analysis................................................................44

5.1 Introduction to Heteroduplex Analysis ......................................................44

5.2 Reagent Preparation................................................................................45

5.3 Gel Volumes ............................................................................................47

5.4 Sample Preparation .................................................................................48

5.5 Temperature Controller............................................................................48

5.6 Adding the Running Buffer .......................................................................49

5.7 Assembling the Heteroduplex Analysis Gel Sandwich .............................50

5.8 Casting Heteroduplex Analysis Gels ........................................................51

Section 6 Single-Stranded Conformational Polymorphism (SSCP) .........52

6.1 Introduction to SSCP ..............................................................................52

6.2 Reagent Preparation ...............................................................................54

6.3 Gel Volumes ............................................................................................56

6.4 Sample Preparation ................................................................................56

6.5 Temperature Controller............................................................................57

6.6 Cooling the Running Buffer and Chiller Settings.......................................58

6.7 Assembling the SSCP Gel Sandwich.......................................................58

6.8 Casting SSCP Gels .................................................................................60

Section 7 Protein Truncation Test (PTT).....................................................61

7.1 Introduction to PTT .................................................................................61

7.2 Reagent Preparation ...............................................................................62

7.3 Gel Volumes ............................................................................................64

7.4 Sample Preparation .................................................................................64

7.5 Temperature Controller............................................................................65

7.6 Adding the Running Buffer .......................................................................66

7.7 Assembling the PTT Gel Sandwich..........................................................66

7.8 Casting PTT Gels ....................................................................................68

Section 8 Electrophoresis...........................................................................69

8.1 Assembling the Upper Buffer Chamber....................................................69

8.2 Sample Loading.......................................................................................71

8.3 Running the Gel.......................................................................................71

8.4 Removing the Gel ....................................................................................73

8.5 Staining and Photographing the Gel ........................................................74

Section 9 Troubleshooting Guide ..............................................................75

9.1 Equipment ..............................................................................................75

9.2 Applications .............................................................................................77

Section 10 Specifications..............................................................................81

Section 11 Maintenance ................................................................................82

Section 12 References...................................................................................82

Section 13 Instruments and Reagents for Mutation Detection

Electrophoresis ..........................................................................83

Section 1 General Safety Information

1.1 Caution Symbols

Read the manual before using the DCode system. For technical assistance, contact your local Bio-Rad Office or in the U.S., call Technical Services at 1-800-4BIORAD (1-800-424-6723). DC power to the DCode system is supplied by an external DC voltage power supply. This power supply must be ground isolated so that the DC voltage output floats with respect to ground. All BioRad power supplies meet this important safety requirement. Regardless of which power supply is used, the maximum specified operating parameters for the system are:

• Maximum voltage limit 500 VDC

• Maximum power limit 50 watts

AC current for controlling temperature to the system, and DC current for electrophoresis, provided by the external power supply, enter the unit through the lid assembly, which provides a safety interlock. DC current to the cell is broken when the lid is removed. Do not attempt to circumvent this safety interlock. Always disconnect the AC cord from the unit and the cord

from the DC power supply before removing the lid, or when working with the cell.

Definition of Symbols

Caution, risk of electric shock.

Caution

Caution, hot surface.

1.2 Precautions During Set-up

• Do not use near flammable materials.

• Always inspect the DCode system for damaged components before use.

• Always place the DCode system on a level bench-top.

• Always place the lid assembly on the buffer tank with the AC and DC power cords

disconnected.

• Always connect the system to the correct AC and DC power sources.

1.3 Precautions During a Run

• Do not run the pump when it is dry. Always add buffer to the “Fill” line when

pre-chilling and/or preheating the buffer; always keep the buffer below “Max” level during

electrophoresis.

• Do not touch any wet surface unless all the electrical sources are disconnected.

• Do not put anything on the top surface of the DCode system module.

1.4 Precautions After a Run

• Always turn off power switches and unplug all cables to DC and AC sources. Allow the

heater tube to cool down (approximately 1 minute) before removing it from the tank. The

ceramic tube may be very hot after shut-down. Do not touch the ceramic tube after turn-

ing off the power.

• Do not cool the hot ceramic tube in cool liquids.

• Always store the electrophoresis/temperature control module on the aluminum DCode

lid stand for maximum stability. Caution: the heater tube may be hot after use

1.5 Safety

This instrument is intended for laboratory use only

This product conforms to the “Class A” standards for electromagnetic emissions intended for laboratory equipment applications. It is possible that emissions from this product may interfere with some sensitive appliances when placed nearby or in the same circuit as those appliances. The user should be aware of this potential and take appropriate measures to avoid interference.

Section 2 Introduction

2.1 Introduction to Mutation Detection Technology

Detecting single base mutations is of utmost importance in the field of molecular genetics. Screening for deletions, insertions, and base substitutions in genes was initially done by Southern blotting. Many techniques have been developed to analyze the presence of mutations in a DNA target. The most common methods include: Single-Strand Conformational Polymorphism

(SSCP), Denaturing Gradient Gel Electrophoresis2(DGGE), carbodiimide3(CDI), Chemical Cleavage of Mismatch4(CCM), RNase cleavage,5Heteroduplex analysis,6and the Protein Truncation Test7(PTT). A new technique for mutation detection is Temporal Temperature Gradient Gel Electrophoresis8(TTGE). Bio-Rad reduced this technique to a simple, reproducible method on the DCode system. TTGE uses a polyacrylamide gel containing a constant concentration of urea. During electrophoresis, the temperature is increased gradually and uniformly. The result is a linear temperature gradient over the course of the electrophoresis run. Many labs used combination of methods to maximize mutation detection efficiency.

The DCode system is a vertical electrophoresis instrument for the detection of gene mutations. The DCode system can be used to perform any vertical gel-based mutation detection method. The system is optimized for DGGE, CDGE, TTGE, SSCP, PTT and Heteroduplex Analysis. Some of the advantages of the DCode system include uniform buffer temperature around the gel, buffer circulation, buffer temperature runs from 5 to 70 °C and a modular design to allow customization.

Section 3 Product Description

3.1 Packing List

The DCode system is shipped with the following components. If items are missing or damaged, contact your local Bio-Rad office.

The DCode System for DGGE (10 cm and 16 cm systems)

Item Quantity

Instruction manual 1 Warranty card (please complete and return) 1 Electrophoresis/temperature control module 1 Electrophoresis tank 1 Casting stand with sponges 1 Sandwich core 1 DCode lid stand 1 16 cm glass plates (16 cm system) 2 sets 10 cm glass plates (10 cm system) 2 sets Sandwich clamps 2 sets Spacers, grooved, 1 mm 2 sets Middle spacer, 1 mm (10 cm system) 2 Prep comb, 1 well, 1 mm (16 cm system) 2 Prep comb, 2 well, 1 mm (10 cm system) 2 16-well comb, 1 mm 1 Comb gasket for 0.75 & 1 mm spacers 1 Comb gasket holder 1 Model 475 gradient former 1 Syringes: 10 ml, 30 ml 2 each Tubing 3 feet Luer couplings 4 Luer syringe locks 2 Syringe sleeves 4 Syringe cap screws 2 Y-fitting 5 Control reagent kit for DGGE, CDGE, TTGE 1

DCode System for CDGE

Item Quantity

Instruction manual 1 Warranty card (please complete and return) 1 Electrophoresis/temperature control module 1 Electrophoresis tank 1 Casting stand with sponges 1 Sandwich core 1 DCode lid stand 1 Glass plates, 16 cm 2 sets Sandwich clamps 2 sets Spacers, 1 mm 2 sets 20-well combs, 1 mm 2 Control reagent kit for DGGE, CDGE, TTGE 1

DCode System for TTGE

Item Quantity

Instruction manual 1 Warranty card (please complete and return) 1 Electrophoresis/temperature control module 1 Electrophoresis tank 1 Casting stand with sponges 1 Sandwich core 1 DCode lid stand 1 Glass plates, 16 cm 2 sets Sandwich clamps 2 sets Spacers, 1 mm 2 sets 20-well combs, 1 mm 2 Control reagent kit for DGGE, CDGE, TTGE 1

DCode System for SSCP

Item Quantity

Instruction manual 1 Warranty card (please complete and return) 1 Electrophoresis/temperature control module 1 Electrophoresis cooling tank 1 Casting stand with sponges 1 Sandwich core 1 DCode lid stand 1 Sandwich clamps 2 sets Glass plates, 20 cm 2 sets Spacers, 0.75 mm 2 sets 20-well combs, 0.75 mm 2 Control reagent kit for SSCP 1

DCode System for Heteroduplex Analysis

Item Quantity

Instruction manual 1 Warranty card (please complete and return) 1 Electrophoresis/temperature control module 1 Electrophoresis tank 1 Casting stand with sponges 1 Sandwich core 1 DCode lid stand 1 Sandwich clamps 2 sets Glass plates, 20 cm 2 sets Spacers, 0.75 mm 2 sets 20-well combs, 0.75 mm 2 Control reagent kit for Heteroduplex Analysis 1

DCode System for Protein Truncation Test

Item Quantity

3.2 System Components and Accessories

Fig. 3.1. The DCode system.

System Components and Accessories Description

Electrophoresis Tank The electrophoresis tank is a reservoir for the running buffer.

Electrophoresis Cooling Tank The electrophoresis cooling tank has two ceramic cooling (SSCP only) fingers inside the electrophoresis tank (Figure 3.2). Two

quick-release connectors are connected to an external chiller to chill the running buffer. The electrophoresis cooling tank should not be used for heated buffer runs (i.e., DGGE, CDGE or TTGE).

Fig. 3.2. Electrophoresis cooling tank.

Model 475 Gradient Former

Electrophoresis/temperature control module

Casting stand with perpendicular assembly

Electrophoresis tank

Core

Casting stand

Ceramic cooling fingers

Fig. 3.3. Electrophoresis/Temperature Control Module.

System Components and Accessories Description

Electrophoresis/Temperature The control module contains the heater, stirrer, pump, Control Module and electrophoresis leads to operate the DCode

system (Figure 3.3). Combined with the lower buffer tank, the control module acts to fully enclose the system. The control module should be placed so that the tip of the stirring bar fits inside the support hole of the tank. The clear loading lid is a removable part that contains four banana jacks which function as a safety interlock. It should be left in place at all times except while loading samples.

Core The sandwich core holds one gel assembly on each side

(Figure 3.4). When attached, each gel assembly forms one side of the upper buffer chamber. The inner plate is clamped against a rubber gasket on the core to provide a greaseless seal for the upper buffer chamber.

Fig. 3.4. Core

Buffer level interlock

Temperature sensor

Buffer recirculation pump

Buffer recirculation port

Ceramic heater

Stirrer

Temperature controller

Casting Stand The casting stand holds the gel sandwich upright

while casting a gel (Figure 3.5). With the cam levers engaged, the sponge seals the bottom of the gel while the acrylamide polymerizes.

Fig. 3.5. Casting Stand.

Sandwich Clamps The sandwich clamps consist of a single screw

mechanism which makes assembly, alignment, and disassembly of the gel sandwich an effortless task. The clamps exert an even pressure over the entire length of the glass plates. A set consists of a left and right clamp.

Alignment Card The alignment card simplifies sandwich assembly by

keeping the spacers in the correct position.

Comb Gasket Holder The comb gasket holder holds the comb gasket that

prevents (DGGE only) leakage of acrylamide during gel casting (Figure 3.6). The front of the holder has two screws which are used to secure the comb gasket against the glass plate. The top of the comb gasket holder also has two tilt rod screws which control the position of the tilt rod during gel casting. The opposite side of the comb gasket holder has two vent ports. There are two sizes of comb gasket holder: a 1 mm size for 1 mm and 0.75 mm spacer sets and a 1.5 mm size for the 1.5 mm spacer set.

Fig. 3.6. Comb gasket holder.

Notched step

Gasket holder

Comb gasket screws

Tilt rod

Stopcocks (DGGE only) The gel solution is introduced via the stopcock at the

inlet port on the sandwich clamp when casting a perpendicular gel (Figure 3.7).

Fig. 3.7. Stopcock

Comb and Spacer Set The 7.5 x 10 cm gel format consist of two “mirror image

(DGGE only)spacers, one middle spacer and a dual prep comb for two 7.5 x 10 cm gels (Figure 3.8). The spacers have a groove and injection port hole for casting. The middle spacer between the two gels fits into the middle notch of the dual comb and allows the air to escape through the comb gasket holder vent port. The 16 x 16 cm gel format consist of two different spacers, one with the groove and injection port hole for casting and one with a short groove toward the injection port hole for air to escape. A single prep comb without a middle spacer is provided with the 16 x 16 cm gel format.

Fig. 3.8. Dual prep comb and spacer set for two 7.5 x 10 cm gels.

Comb and Spacer Set Different types of combs and spacers are provided with

the different DCode systems. Spacer lengths are 10 cm, 16 cm or 20 cm, with a thickness of 0.75 or

1.0 mm. Combs come with 16 or 20 wells and a thickness of 0.75 or 1.0 mm.

Inlet fitting

Injection port holes

groove

Fig. 3.9. Pressure clamp.

Pressure Clamp The pressure clamp provides consistent pressure to the (DGGE only) comb gasket before securing it to the plate assembly

to provide a seal (Figure 3.9).

DCode Lid Stand The DCode lid stand provides a stand when the

electrophoresis/temperature control module (lid) is not in use. The stand must be used to properly support and protect the lid components when the lid is not on the electrophoresis tank.

Fig. 3.10. Model 475 Gradient Delivery System.

Gasket holder

Comb gasket

Air vent

Pressure clamp

Pressure clamp screw

Large glass plate

Small glass plate

Luer fitting

VOLUME 10 ML SYRINGE

MODEL 475 GRADIENT DELIVERY SYSTEM

THIS SIDE FOR HIGH DENSITY SOLUTION (BOTTOM FILLING)

LOW DENSITY SOLUTION (TOP FILLING)

Syringe holder

DELIVER

Cam

Plunger cap screw

Plunger cap

Lever

Y-Fitting

Tubing

Syringe

Syringe sleeve

Syringe holder screw

Volume setting indicator

Volume adjustment screw

1234 5 6 78910

5 6

Model 475 System Components

Description (DGGE System only)

Syringe Two pairs each of 10 and 30 ml disposable syringes are

provided. The 10 ml syringes are for gel volumes less than 10 ml. The 30 ml syringes are for gel volumes greater than 10 ml. For proper gel casting, use matching syringe sizes.

Plunger Caps/ There are two plunger caps, one for each syringe. The Plunger Cap Screw plunger caps fit both the 10 and 30 ml syringes (Figure 3.11).

Lever Attachment Screw The lever attachment screw is on the plunger cap. This

screw fits into the groove of the lever and conducts the driving force of the cam in dispensing the gel solution.

Syringe Sleeve One pair of syringe sleeves for each size syringe is

provided (Figure 3.12). The sleeve is a movable adaptor to mount the syringe in the holder. The sleeve should conform to the syringe. If the syringe is too tight or too loose, adjust the sleeve by pushing or pulling.

Syringe Holder/ The syringe holder is next to the lever. It holds the syringe Syringe Holder Screw in place and controls the delivery volume. The syringe is held

in the holder by tightening the holder screw against the sleeve.

Volume Adjustment Screw The volume adjustment screw is on both sides of the syringe

holder (Figure 3.10). It adjusts the holder to the desired delivery volume.

Volume Setting Indicator The volume setting indicator is at the top corner of the syringe

holder nearest the volume setting numbers (Figure 3.10).

Lever The position of the lever is controlled by the rotation of the

cam (Figure 3.10). The lever must be in the vertical or start position before use.

Tygon Tubing One length of Tygon™tubing is provided. Cut the tubing into two

15.5 cm and one 9 cm lengths. The longer pieces are used to transport the gel solution from the syringes into the Y-fitting. The short piece will transport the gel solution from the Y-fitting to the gel sandwich.

Y-Fitting The Y-fitting mixes the high and low density gel

solutions (Figure 3.13).

Luer Fitting/Coupling There are two luer fittings that fit both 10 and 30 ml syringes.

The fittings twist onto the syringe and connect to the Tygon tubing on the other end. A luer coupling is used on one end of the 9 cm tubing to connect it to the gel sandwich stopcock.

Fig. 3.11. Plunger Cap Fig. 3.12. Syringe sleeve Fig. 3.13. Y-Fitting

Lever attachment screw

Plunger cap screw

Section 4 Denaturing Gel Electrophoresis (DGGE, CDGE, TTGE)

4.1 Introduction to Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis (DGGE) is an electrophoretic method to identify single base changes in a segment of DNA. Separation techniques on which DGGE is based were first described by Fischer and Lerman.

In a denaturing gradient acrylamide gel, double-stranded DNA is subjected to an increasing denaturant environment and will melt in discrete segments called "melting domains". The melting temperature (Tm) of these domains is sequence-specific. When the Tmof the lowest melting domain is reached, the DNA will become partially melted, creating branched molecules. Partial melting of the DNA reduces its mobility in a polyacrylamide gel. Since the Tmof a particular melting domain is sequence-specific, the presence of a mutation will alter the melting profile of that DNA when compared to wild-type. DNA containing mutations will encounter mobility shifts at different positions in the gel than the wild-type. If the fragment completely denatures, then migration again becomes a function of size (Figure 4.1).

Fig. 4.1. An example of DNA melting properties in a perpendicular denaturing gradient gel. At a low concentration of denaturant, the DNA fragment remains double-stranded, but as the concentration of denaturant increases, the DNA fragment begins to melt. Then, at very high concentrations of denaturant, the DNA fragment can completely melt, creating two single strands.

In DGGE, the denaturing environment is created by a combination of uniform temperature, typically between 50 and 65 °C and a linear denaturant gradient formed with urea and formamide. A solution of 100% chemical denaturant consists of 7 M urea and 40% formamide. The denaturing gradient may be formed perpendicular or parallel to the direction of electrophoresis. A perpendicular gradient gel, in which the gradient is perpendicular to the electric field, typically uses a broad denaturing gradient range, such as 0–100% or 20–70%.

In parallel DGGE, the denaturing gradient is parallel to the electric field, and the range of denaturant is narrowed to allow better separation of fragments.9Examples of perpendicular and parallel denaturing gradient gels with homoduplex and heteroduplex fragments are shown in Figure 4.2.

Double strand

Single strands

Denaturant

Wild Type

Mutant

100%0%

Partially melted "wild type"

Partially melted "mutant"

Electrophoresis

Denaturant

Partially melted “mutant”

Partially melted “wild type”

100%

Electrophoresis

Single strands

Wild Type

Mutant

Double strand

Fig. 4.2. A. Perpendicular denaturing gradient gel in which the denaturing gradient is perpendicular to the electrophoresis direction. Mutant and wild-type alleles of exon 6 from the TP53 gene amplified from primary breast

carcinomas and separated by perpendicular DGGE (0–70% denaturant) run at 80 V for 2 hours at 56 °C. The first two bands on the left are heteroduplexes and the other two bands are the homoduplexes. B. Parallel denaturing gradient gel in which the denaturing gradient is parallel to the electrophoresis direction. Mutant and wild-type alleles of exon 8 from the p53 gene separated by an 8% acrylamide:bis (37.5:1) gel with a parallel gradient of 40–65% denaturant. The gel was run at 150 V for 2.5 hours at 60 °C in 1x TAE buffer. Lane 1 contains the mutant fragment, lane 2 contains the wild-type fragment, lane 3 contains both the mutant and wild-type fragments.

When running a denaturing gradient gel, both the mutant and wild-type DNA fragments are run on the same gel. This way, mutations are detected by differential migration of mutant and wild-type DNA. The mutant and wild-type fragments are typically amplified by the polymerase chain reaction (PCR) to make enough DNA to load on the gel. Optimal resolution is attained when the molecules do not completely denature and the region screened is in the lowest melting domain. The addition of a 30–40 base pair GC clamp to one of the PCR primers insures that the region screened is in the lower melting domain and that the DNA will remain partially double-stranded.34An alternative to GC clamps is using psoralen derivative PCR primers called ChemiClamp primers.10Because ChemiClamps covalently link the two DNA strands at one end, they should not be used when isolating a DNA fragment which is going to be sequenced from a gel. The size of the DNA fragments run on a denaturing gel can be as large as 1 kb in length, but only the lower melting domains will be available for mutation analysis. For complete analysis of fragments over 1 kb in length, more than one PCR reaction should be performed.

The thermodynamics of the transition of double-stranded to single-stranded DNA have been described by a computer program developed by Lerman.12Bio-Rad offers a Macintosh

computer program, MacMelt™software, which calculates and graphs theoretical DNA melting profiles. Melting profile programs can show regions of theoretical high and low melting domains of a known sequence. Placement of primers and GC clamps can be optimized by analysis of placement effect on the DNA melting profile.

The method of creating heteroduplex molecules helps in detecting homoduplex mutations. This process is typically done when it is not originally possible to resolve a homoduplex mutation. Analysis of heteroduplex molecules can, therefore, increase the sensitivity of DGGE. Heteroduplexes can be formed by adding the wild-type and mutant template DNAs in the same PCR reaction or by adding separate PCR products together, then denaturing and allowing them to re-anneal. A heteroduplex has a mismatch in the double-strand causing a distortion in its usual conformation; this has a destabilizing effect and causes the DNA strands to denature at a lower concentration of denaturant (Figure 4.3). The heteroduplex bands always migrate more slowly than the corresponding homoduplex bands under specific conditions.

40%

70%

65%

Fig. 4.3. An example of wild-type and mutant DNA fragments that were denatured and re-annealed to generate four fragments: two heteroduplexes and two homoduplexes run on a parallel denaturing gradient gel. The melting behavior of the heteroduplexes is altered so that they melt at a lower denaturant

concentration than the homoduplexes and can be visualized on a denaturing gradient gel.

Reagent Preparation

The concentration of denaturant to use varies for the sample being analyzed with the DCode system. Typically a broad denaturing gradient range is used, such as 0–100% or 20–70%. The concentration of acrylamide can also vary, depending on the size of the fragment analyzed. Both 0% and 100% denaturant should be made as stock solutions. A 100% denaturant is a mixture of 7 M urea and 40% deionized formamide. Reagents for casting and running a DGGE gel are included in the DCode Electrophoresis Reagent Kit for DGGE/CDGE, catalog number 170-9032.

For different percent crosslinking, use the equation below to determine the amount of Bis to add. The example stock solution below is for an acrylamide/bis ratio of 37.5:1.

40% Acrylamide/Bis (37.5:1) Reagent Amount

Acrylamide 38.93 g Bis-acrylamide 1.07 g dH2O to 100.0 ml

Filter through a 0.45 µ filter and store at 4°C.

Wild Type DNA Mutant DNA

Wild-Type DNA

Mutant DNA

wt + mut

20%

60%

mut

Heteroduplex

DNA

Homoduplex

DNA

Homoduplexes

Heteroduplexes

Denature and reanneal

Denaturant

Denature and reanneal

Homoduplex

DNA

wt mut wt + mut

Heteroduplex

DNA

Heteroduplexes

Homoduplexes

Polyacrylamide gels are described by reference to two characteristics:

1) The total monomer concentration (%T)

2) The crosslinking monomer concentration (%C)

%T =

gm acrylamide + gm bis-acrylamide

x 100

Total Volume

%C =

gm bis-acrylamide

x 100

gm acrylamide + gm bis-acrylamide

50x TAE Buffer Reagent Amount Final Concentration

Tris base 242.0 g 2 M Acetic acid, glacial 57.1 ml 1 M

0.5 M EDTA, pH 8.0 100.0 ml 50 mM dH2O to 1,000.0 ml

Mix. Autoclave for 20–30 minutes. Store at room temperature.

The table below provides the percentage acrylamide/bis needed for a particular size range.

Gel Percentage Base Pair Separation

6% 300–1000 bp 8% 200–400 bp

10% 100–300 bp

0% Denaturing Solution

6% Gel 8% Gel 10% Gel

12% Gel

40% Acrylamide/Bis 15 ml 20 ml 25 ml 30 ml 50x TAE buffer 2 ml 2 ml 2 ml 2 ml dH2O 83 ml 78 ml 73 ml 68 ml Total volume 100 ml 100 ml 100 ml 100 ml

Degas for 10–15 minutes. Filter through a 0.45 µ filter. Store at 4°C in a brown bottle for approximately 1 month.

100% Denaturing Solution

6% Gel 8% Gel

10% Gel 12% Gel

40% Acrylamide/Bis 15 ml 20 ml 25 ml 30 ml 50x TAE buffer 2 ml 2 ml 2 ml 2 ml Formamide (deionized) 40 ml 40 ml 40 ml 40 ml Urea 42 g 42 g 42 g 42 g dH2O to 100 ml to 100 ml to 100 ml to 100 ml

Degas for 10–15 minutes. Filter through a 0.45 µ filter. Store at 4°C in a brown bottle for approximately 1 month. A 100% denaturant solution requires re-dissolving after storage. Place the bottle in a warm bath and stir for faster results.

For denaturing solutions less than 100%, use the volumes for acrylamide, TAE and water described above in the 100% Denaturing Solution. Use the amounts indicated below for urea and formamide.

Denaturing Solution 10% 20% 30% 40% 50% 60% 70% 80% 90%

Formamide (ml) 4 8 12 16 20 24 28 32 36 Urea (g) 4.2 8.4 12.6 16.8 21 25.2 29.4 33.6 37.8

10% Ammonium Persulfate Reagent Amount

Ammonium persulfate 0.1 g dH2O 1.0 ml

Store at –20°C for about a week.

DCode Dye Solution Reagent Amount Final Concentration

Bromophenol blue 0.05 g 0.5% Xylene cyanol 0.05 g 0.5% 1x TAE buffer 10.0 ml 1x

Store at room temperature. This reagent is supplied in the DCode electrophoresis reagent kit for DGGE, CDGE.

2x Gel Loading Dye Reagent Amount Final Concentration

2% Bromophenol blue 0.25 ml 0.05% 2% Xylene cyanol 0.25 ml

0.05% 100% Glycerol 7.0 ml 70% dH2O 2.5 ml Total volume 10.0 ml

Store at room temperature.

1x TAE Running Buffer Reagent Amount

50x TAE buffer 140 ml dH2O 6,860 ml Total volume 7,000 ml

Gel Volumes

Linear Denaturing Gradient Gels

The table below provides the required gradient delivery system setting per gel size desired. The volume per syringe is for the amount required for each low and high density syringe, and the volume adjustment setting sets the gradient delivery system for the proper delivery of solutions. The 7.5 x 10 cm and 16 x 16 cm size gels are recommended for the perpendicular gel formats, whereas the 16 x 10 cm and 16 x 16 cm gel formats are recommended for parallel denaturing gels. The volume per syringe requires a larger volume of reagent than the volume setting indicates, because the excess volume in the syringe is needed to push the correct amount of gel solution into the gel sandwich.

Volume Volume per Adjustment

Spacer Size Gel Size Syringe Setting

0.75 mm 7.5 x 10 cm 5 ml 3.5

16 x 10 cm 8 ml 6.5 16 x 16 cm 11 ml 9.5

1.00 mm 7.5 x 10 cm 6 ml 4.5

16 x 10 cm 11 ml 9.5 16 x 16 cm 16 ml 14.5

1.50 mm 7.5 x 10 cm 8 ml 6.5

16 x 10 cm 15 ml 13.5 16 x 16 cm 24 ml 22.5

Spacer Thickness 16 x 16 cm Gel 16 x 10 cm Gel

0.75 mm 25 ml 15 ml

1.00 mm 30 ml 20 ml

1.50 mm 45 ml 26 ml

Sample Preparation

1. It is important to optimize the PCR reaction to minimize unwanted products which may

interfere with gel analysis. PCR products should be evaluated for purity by agarose gel

electrophoresis before being loaded onto a denaturing acrylamide gel.

2. For a perpendicular denaturing gel, load about 1–3 µg of amplified DNA per well

(usually 50% of a 100 µl PCR volume from a 100 ng DNA template). Both wild-type and

mutant samples are mixed together and run on the same gel.

3. For a parallel denaturing gel, load 180–300 ng of amplified DNA per well (usually 5–10%

of a 100 µl PCR volume from a 100 ng DNA template). A wild-type control should be run

on every gel.

4. Add an equal volume of 2x gel loading dye to the sample.

5. Heteroduplexes can be generated during PCR by amplifying the mutant and wild-type samples

in the same tube. If the samples are amplified in separate tubes, then heteroduplexes can be

formed by mixing an equal amount of mutant and wild-type samples in one tube. Heat the tube

at 95 °C for 5 minutes, then place at 65°C for 1 hour, and let slowly cool to room temperature.

Temperature Controller

The temperature controller maintains the desired buffer temperature in the DCode system.

Turning on the Controller

Turn the controller on by pressing the power button and wait for the controller to initialize. The controller will display the following screens in order:

• The program code

• The date code and serial code

• The hours used

• The temperature control screen (see Fig. 1).

The controller is now ready to be programmed.

Setting the Set Temperature and Ramp Rate

1. From the temperature control screen, use the arrow keys to adjust the Set Temperature to your desired target temperature in °C (Fig. 1)

2. Use the key to display the ramp control screen. (Fig. 2.)

3. From the ramp control screen, use the arrow keys to set the desired temperature Ramp Rate in °C per hour. Note that the maximum ramp rate for the DCode is 50°C /hr. If the target Ramp Rate is set using the controller to higher than 50°C /hr the maximum ramp rate of 50°C /hr will be used.

4. To toggle between the temperature control screen and the ramp control screen, or to review the set temperature and ramp rate, press the key at any time

Note: Following programming by default the controller displays the Step Temperature rather than the Set Temperature (see Fig 1. below). The step temperature is the next temperature value the system will reach as it heats/cools to reach set temperature. To display the Set Temperature press the key.

Fig. 1. Temperature Control Screen.

Fig. 2. Ramp Control Screen.

Pre-heating the Running Buffer

1. Fill the electrophoresis tank with 7 L of 1x TAE running buffer.

Note: It is recommended that the running buffer not be reused. Reusing the running buffer may affect the migration rate and band resolution.

2. Place the temperature control module on top of the electrophoresis tank. Attach the power cord to the temperature control module and turn the power, pump, and heater on. The clear loading lid should be on the temperature control module during preheating.

3. Set the temperature controller to the desired temperature. Set the temperature ramp rate to 200 °C/hr to allow the buffer to reach the desired temperature the quickest.

4. Preheat the buffer to the set temperature. It can take 1 to 1.5 hours for the system to heat the buffer up to the set temperature. Heating the buffer in a microwave helps reduce the preheating time.

Assembling the Perpendicular Gradient Gel Sandwich

For perpendicular gel formats, 7.5 x 10 cm (dual) and 16 x 16 cm (single) gel sandwich sizes

are recommended. These two different perpendicular gel formats consist of a set of spacers that provide casting at the side of the gel sandwich via the stopcock. To insure proper alignment, make sure all plates and spacers are clean and dry before assembly. Use caution when assembling the glass plate sandwiches. Wear gloves and eye protection at all times.

1. Assemble the gel sandwich on a clean surface. Lay the large rectangular plate down first, then place the left and right spacers of equal thickness along the short edges of the larger rectangular plate. To assemble perpendicular gradient gels, place the spacers so that the holes on the spacers are at the top of the plate with the grooved side of the spacer against the larger glass plate. When properly placed, the notched edges of the spacers will face each another.

2. Place a short glass plate on top of the spacers so that it is flush with the bottom edge of the long plate.

3. Loosen the single screw of each sandwich clamp by turning counterclockwise. Place each clamp by the appropriate side of the gel sandwich with the locating arrows facing up and toward the glass plates (Figure 4.5).

Fig. 4.5. Positioning glass plates, spacers, and clamps.

4. Grasp the gel sandwich firmly. Guide the left and right clamps onto the sandwich so that the long and short plates fit the appropriate notches in the clamp (Figure 4.6). Tighten the screws enough to hold the plates in place.

Fig. 4.6. Attaching the clamps to the glass plate assembly.

5. Place the sandwich assembly in the alignment slot (the slot without cams) of the casting stand with the short glass plate forward (Figure 4.7). Loosen the sandwich clamps and insert an alignment card to keep the spacers parallel to the clamps.

Note: Always use the alignment slot and alignment card to set the spacers in place. Failure to use these can result in gel leakage when casting, as well as buffer leakage during the run.

Fig. 4.7. Aligning spacers in the sandwich assembly.

6. Align the plates and spacers by simultaneously pushing inward on both clamps at the locating arrows while, at the same time, pushing down on the spacers with your thumbs. Tighten both clamps just enough to hold the sandwich in place. Pushing inward on both clamps at the locating arrows will insure that the spacers and glass plates are flush against the sides of the clamps (Figure 4.7).

7. Remove the alignment card. Then, remove the sandwich assembly from the casting stand and check that the plates and spacers are flush at the bottom. If they are not flush, realign the sandwich and spacers (Repeat steps 5–7).

8. When a good alignment and seal are obtained, tighten the clamp screws until it is finger-tight.

9. Place the proper comb in the sandwich and align it against the notches in the spacers. For the 7.5 x 10 cm perpendicular gradient gel, insert the middle spacer into the center of the sandwich until it fits into the middle notch on the comb. Straighten the spacer and the comb. The bottom of the middle spacer should also be flush against the glass plates (Figure 4.8).

Note: The proper comb for a 7.5 x 10 cm gel is the dual comb that requires a middle spacer to separate the two 7.5 x 10 cm gels, whereas the 16 x 16 cm gel comb is a single comb that does not require a middle spacer.

Fig. 4.8. Positioning the middle spacer in a 7.5 x 10 cm gel sandwich assembly.

10. Inspect the comb gasket to insure that the comb gasket is free of gel material. Remove any polymerized material in the comb gasket vent ports. The soft comb gasket should lay flat within the comb gasket holder.

Note: To remove the soft comb gasket from the holder, push the gasket away from the four holes in the holder. To replace the comb gasket, insert the gasket into the holder with the thick portion in first. Place one corner of the gasket against the top portion of the holder. With a flat spatula, guide the four tabs into the four holes. Carefully run the spatula across the gasket to completely set the gasket in place.

11. Stand the sandwich assembly upright on a flat surface. Loosen the comb gasket holder screws until the threads can no longer be seen. Mark an arrow on the middle of the screw head using a permanent marker (this will be the marker for adjusting the proper screw tension). With the comb gasket screws and the long plate facing you, slide the comb gasket holder down over the top of the assembled glass sandwich. When the comb gasket is properly placed, the angled cuts on the edges of the comb gasket will rest on the complementary angled cuts at the top of each spacer. Turn the screws until they just make contact with the glass plate, then twist the screws an additional 1/4 turn.

12. Before the screws can be completely tightened, the pressure clamp must be attached to the sandwich assembly. Loosen the pressure clamp screw. Mark an arrow on the middle of the screw head using a permanent marker (this will be the marker for adjusting the proper screw tension). Lay the pressure clamp on a flat surface so that the notched cut-out faces the ceiling and the pressure clamp screw points up and away from you.

13. Without touching the comb gasket holder, turn the assembled glass sandwich so that the comb gasket screws are facing down and the vent ports are facing up. Center the assembly over the pressure clamp and allow the assembly to rest on top of it. A properly placed pressure clamp will be situated on the middle of the sandwich with the notched cut-out against the bottom of the glass plate. Proper placement of the sandwich assembly in the pressure clamp will insure that equal force is applied to the comb gasket holder during the final tightening of the screws. Twist the pressure clamp screw until it makes contact with the comb gasket holder, then tighten the pressure clamp screw two additional turns (use the arrow to keep track of the turns).

Note: Check to insure that the bottom of the glass plates are still flush. If the plates are off-set, one or both of the sandwich clamps may not be tightened. Repeat steps 5–13.

14. Tighten the comb gasket screws an additional one turn. If it is tightened more, the glass plates may crack. For a proper seal, check to see that the notches on both the comb gasket and spacers are sealed against each other. It is important that the gasket is placed properly to prevent leakage while casting. Remove the pressure clamp.

15. Twist the injection port fitting into the holes on the sandwich clamps. Do not over-tighten; it will damage the O-ring and cause leakage. A snug fit is all that is needed to place the injection port against the glass plate assembly. Push a stopcock into each of the injection port fittings. Make sure that the fit is snug. A loose stopcock may cause leakage during casting.

16. Place the gray sponge onto the front casting slot. The camshafts on the casting stand should have the handles pointing up and pulled out. Place the sandwich on the sponge with the shorter plate facing forward. When the sandwich is placed correctly, press down on the sandwich and turn the handles of the camshaft down so that the cams lock the sandwich in place.

a. For a 7.5 x 10 cm dual gel sandwich, only half of the sandwich is cast at a time. Open

the stopcock and unplug the vent port on the side of the sandwich where the gel is being cast. To prevent leakage the other half of the sandwich should have a closed stopcock and plugged vent port.

b. For a 16 x 16 cm gel sandwich, both vent ports should be plugged to prevent leakage

during casting. The 16 x 16 cm spacers are one orientation only, thus the special casting groove is always on the right-hand side and the smaller, shorter groove on the left-hand side of the gel sandwich.

17. Tilt the gel sandwich assembly and casting stand using the tilt rod as a leg. Adjust the tilt level to the highest etched line on the rod (the one farthest from the black tilt-rod cap) for the 7.5 x 10 cm format and the lowest etched line on the rod for the 16 x 16 cm format.

18. Familiarize yourself with the Model 475 Gradient Delivery System before casting perpendicular gradient gels.

Casting Perpendicular Denaturing Gradient Gels

1. One length of Tygon tubing is provided and should be cut into two 15.5 cm lengths and one 9 cm length. The longer pieces of Tygon tubing will be used to conduct the gel solution from the syringes into the Y-fitting. The short piece of Tygon tubing will conduct the gel solution from the Y-fitting to the gel sandwich. Connect one end of the 9 cm Tygon tubing to the Y-fitting and connect a luer coupling to the other end of the 9 cm tubing. Connect luer fittings onto the two long pieces of tubing. Connect the luer fittings to 10 ml or 30 ml syringes. Do not connect the long Tygon tubing to the Y-fitting at this time.

2. Label one of the syringes LO (for the low density solution) and one HI (for the high density solution). Attach a plunger cap onto each syringe plunger “head.” Position the plunger "head" in the middle of the plunger cap and tighten enough to hold the plunger in place. Position the cap in the middle for proper alignment with the lever on the gradient delivery system. Slide each syringe into a syringe sleeve. Move the sleeve to the middle of the syringe, keeping the volume gradations visible. Make sure that the lever attachment screw is in the same plane as the flat or back side of the sleeve. This is very important for proper attachment of the syringe to the lever.

Note: Insure that the tubing is free of any gel material by pushing water through the tubing with the syringe. The tubing should be free of material before casting, remove any remaining water from the tubing.

3. Rotate the cam wheel counterclockwise to the vertical or start position. To set the desired delivery volume, loosen the volume adjustment screw. Place the volume setting indicator located on the syringe holder to the desired volume setting. Tighten the volume adjustment screw. For 7.5 x 10 cm gels (1 mm thick), set the volume setting indicator to 4.5. For 16 x 16 cm gels (1 mm thick), set the volume setting indicator to 14.5. Refer to Section 4.1.

4. From the stocks solutions, pipet out the desired amounts of the high and low density gel solutions into two disposable test tubes, Section 4.1.

Optional: To visually check the formation of the gradient, add 100 µl of DCode dye solution per 5 ml high density solution.

Note: The gel solution volume should be greater than the amount set on the volume adjustment lever. For example, if the setting indicator is set at 4.5, the syringe should contain 5 ml of the gel solution. This extra solution is needed to push the correct amount into the gel sandwich.

The steps below are time-sensitive (about 7–10 minutes). Insure that steps 1 through 4 are done before proceeding further. Be thoroughly familiar with the following steps before casting the gel.

5. Add a final concentration of 0.09% (v/v) each of ammonium persulfate and TEMED solutions. The 0.09% (v/v) concentrations allow about 5–7 minutes to finish casting the gel before polymerization. Cap and mix by inverting several times. With the syringe connected to the tubing, withdraw all of the high density solution into the HI syringe. Do the same for the low density solution into the LO syringe.

Note: Acrylamide is a very hazardous substance. Use caution: wear gloves and eye protection at all times. Avoid skin contact.

6. Carefully remove air bubbles from the LO syringe by turning it upside down (plunger cap towards the bench) and gently tapping. Push the gel solution to the end of the tubing. Do not push it out of the tubing as loss of gel solution will disturb the volume required to cast the desired gel.

7. Place the LO syringe into the gradient delivery system syringe holder (LO density side) by holding the syringe by the plunger and inserting the lever attachment screw into the lever groove. Do not handle the syringe. It will dispense the gel solution out of the syringe. Casting a perpendicular gel is referred to as a bottom filling method, so place the LO syringe on the correct side of the gradient system.

8. Carefully remove the air bubbles from the HI syringe by turning it upside down (plunger cap towards the bench) and gently tapping. Push the gel solution to the end of the tubing. Do not push it out of the tubing as loss of gel solution will disturb the volume required to cast the desired gel.

9. Place the HI syringe into the gradient delivery system syringe holder (HI density side) by holding the syringe by the plunger and inserting the lever attachment screw into the lever groove. Do not handle the syringe. It will dispense the gel solution out of the syringe.

10. Slide the tubing from the low density syringe to one end of the Y-fitting. Do the same for the high density syringe.

11. Connect the 9 cm tubing with the luer coupling on the sandwich assembly stopcock. Insure that the stopcock is open and that the vent port is unplugged for the half of the sandwich being cast.

Note: For a 16 x 16 cm single gel, both stopcocks are open during casting. After casting, both stopcocks are closed.

12. Rotate the cam wheel slowly and steadily to deliver the gel solution. It is important to cast the gel solution at a steady pace to avoid disturbances between gel solutions within the sandwich.

Fig. 4.9. Casting a perpendicular gradient gel using the Model 475 gradient delivery system.

13. Plug the vent port and close the stopcock on the gel sandwich when the cam wheel has reached the stop position. Carefully level the gel sandwich by adjusting the gasket tilt rod. Be sure to loosen the tilt rod screw and not the sandwich clamp screw.

Note: For a properly cast perpendicular gradient gel it is extremely important to level the sandwich assembly after casting.

14. Immediately remove the tubing from the sandwich assembly stopcock. Place the tubing into a beaker of water and reverse the cam on the Gradient Delivery System. This will rinse the tubing and Y-fitting. Remove both syringes from the syringe holder on the gradient delivery system. Detach the syringe tubing from the Y-fitting. Run or push water out through the syringe, tubing and Y-fitting several times to get rid of any residual gel solution. It is extremely important that this is done quickly after casting to avoid any gel polymerization.

15. Let the gel polymerize for about 60 minutes. To cast the other half of the 7.5 x 10 cm gel format, remove the gasket tilt rod and place it on the other side of the comb gasket. Repeat steps 4 through 15.

Note: If casting a single 7.5 x 10 cm gel, let the gel solution polymerize for 60 minutes. Carefully remove the comb gasket; leave the comb in place and pipette (on an opening near the spacer) a 10 ml gel solution plus initiators in the uncast half of the sandwich to create a dam.

16. After polymerization, remove the comb by pulling it straight up slowly and gently.

17. Continue with Section 8 for electrophoresis.

Assembling the Parallel Gradient Gel Sandwich

For parallel gel formats, a 16 x 16 cm gel sandwich size is recommended. The parallel gel

format does not require special casting grooves in the spacers, so the straight edge portion (ungrooved side) of the spacers is used. To insure proper alignment, make sure all plates and spacers are clean and dry before assembly. Use caution when assembling the glass plate sandwiches. Wear gloves and eye protection at all times.

1. Assemble the gel sandwich on a clean surface. Lay the large rectangular plate down first, then place the left and right spacers of equal thickness along the short edges of the larger rectangular plate. To assemble parallel gradient gels, place the spacers so that the grooved opening of the spacers face the sandwich clamps. When properly placed, the grooved side of the spacers and the notches will face the sandwich clamps, and the hole is located near the top of the plates.

2. Place the short glass plate on top of the spacers so that it is flush with the bottom edge of the long plate.

3. Loosen the single screw of each sandwich clamp by turning each screw counterclockwise. Place each clamp by the appropriate side of the gel sandwich with the locating arrows facing up and toward the glass plates (Figure 4.10).

Fig. 4.10. Positioning glass plates, spacers, and clamps.

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