Extraction of nucleic acids          *

Introduction     *

DNA Extraction From Whole Blood      *

DNA Extraction Using A Variation Of The Puregene Method (Salt precipitation)        *

Method            *

Phenol-Chloroform Extraction *

Method            *

DNA extraction from buccal samples (Qiagen system)         *

DNA extraction from Wax Blocks        *

DNA Concentration Assays     *

Solutions          *

Equipment       *

Method            *

Assessing the purity of DNA     *

OD260/280 ratios         *

Gel analysis of DNA     *

Polymerase chain reaction for the analysis of genomic DNA.          *

Reagents         *

Equipment       *

PCR Machines (Thermal Cyclers)         *

Hot start PCR protocol using heat-activated polymerase     *

Stochiometry of the PCR          *

(after Ruano G, Brash DE, Kidd KK, 1991 Amplifications (7) p1-4.)       *

ARMS:           *

CF (12)m - Elucigene ARMS PCR Kit     *

Materials          *

Method            *

Results  *

DASH (Dynamic Allele Specific Hybridisation)          *

Protocol and Troubleshooting Guide  *

DASH Recommendations        *

DASH PCR Amplification Protocol       *

PCR Conditions            *

PCR Set Up      *

Run the PCR    *

The DASH Assay          *

Binding Product to Plate and Hybrid Preparation      *

Reading a DASH Plate *

Manual SSCP and heteroduplex gels   *

Equipment       *

Solutions          *

other materials            *

GEL PREPARATION       *

8% SSCP GEL    *

8% HETERODUPLEX GELS           *

Samples, preparation and electrophoresis    *

Silver staining of polyacrylamide gels *

Guide to interpretation           *

Troubleshooting guide            *

Diagnostic testing for breast / ovarian cancer using SSCP/Heteroduplex analysis followed by protein trunction testing.            *

Mutation Analysis       *

Multiplex Heteroduplex Analysis           *

PTT Analysis       *

BRCA2 Exon 11 PTT Analysis    *

Denaturing Gradient Gel Electrophoresis       *

1. Assembly of the electrophoresis cassette:   *

2. Preparing the cassette for perpendicular electrophoresis:  *

3. Casting a gel in the INGENYphorU-2           *

4.running a gel:           *

5. Gel handling after electrophoresis: *

Step by step manual for the INGENYphorU buffer system       *

BRCA1 and BRCA2 DGGE        *

Amplification   *

DGGE  *

Ethidium bromide staining of polyacrylamide gels     *

Silver staining of polyacrylamide gels *

Solutions          *

Method            *

Denaturing gradient gel electrophoresis (DGGE) protocol    *

Milan Macek M.D. (Hum Mutation 9: 136 1997)           *

Protein truncation test      *

Radioactive Protein Truncation Test (PTT) From Genomic DNA - introduction            *

Storage of Reagents   *

SOLUTIONS        *

Polymerase Chain Reaction for PTT    *

PTT Reactions   *

Electrophoresis            *

Gel Fixing         *

Radioactive Enhancement     *

Autoradiography         *

References       *

DHPLC          *

Structure          *

Background    *

Sample Preparation for DHPLC           *

Interpretation of DHPLC data *

Troubleshooting guide.           *

No sample peak in the chromatogram          *

Large spikes in the middle of the chromatogram      *

Small spikes on the acetonitrile wash off peak          *

Sample peak eluting on the acetonitrile wash off peak        *

Large broad peak eluting at about 2-3 minutes.       *

FLUORESCENT SSCP ANALYSIS        *

SOLUTIONS       *

GEL PREPARATION       *

6.5% SSCP Acrylamide GEL      *

MDE SSCP GEL *

Casting the gel           *

SSCP SAMPLE PREPARATION     *

Setting up the 377 Computer for an SSCP run *

Before the run: *

Run Control Panel       *

Gel’s Matrix File:           *

Sample Sheet: *

To make a new sample sheet:            *

To Amend a Sample Sheet      *

Setting up the 377 for an SSCP Run:    *

Equipment Start-up     *

Status of 377    *

Plate Check    *

Pre-run *

Automated Sequencing using the 377 *

Overview         *

Solutions          *

Stock Acrylamide solution       *

To prepare 250ml Acrylamide for 5 gel runs using 36cm plates           *

PCR solutions   *

Agarose gel solutions  *

Big Dye Terminator Mix            *

Half-Term (Big Dye)      *

Primers *

95% Ethanol     *

70% Ethanol     *

Loading Buffer *

Method            *

Casting the gel           *

Template preparation *

Sequencing reaction   *

Purification of sequencing reactions   *

Electrophoresis            *

Data handling and QA           *

ABI PRISMR BigDyeTM Terminator Cycle Sequencing          *

Cycle Sequencing on the GeneAmp 9600 or 2400:     *

Purifying the Extension Products         *

Ethanol/Sodium Acetate Precipitation:          *

Electrophoresis on the ABI PRISM 310   *

Minisequencing      *

Mini sequencing protocol using SnaPshot Kit  *

Solutions          *

Method            *

Template purifiation    *

Gene dosage by semi-quantitative PCR          *

Principle           *

Method            *

Table 1 Primer sequences        *

Figure 1 Multiplex Amplicons   *

Table 2 Dosage quotients       *

References       *

Single Nucleotide Polymorphisms (SNPs) using electronic hybridization        *

Introduction     *

Materials Required     *

Solutions and Reagents          *

Equipment       *

Solution Preparation   *

SNP Protocols   *

Amplicon and Oligo Design for SNP Applications     *

SNP Components        *

Amplicon Sample Preparation           *

Running SNP Protocols on the Nanochip Molecular Biology Workstation      *

SNP Amplicon-Down Amplicon/Stabilizer Oligo Address      *

SNP Capture-Down Stabilizer Oligo and Amplicon Address *

SNP Reporter Hybridization     *

SNP Thermal Discrimination    *

Enzyme mismatch detection      *

EMC assay protocols   *

Chemical Cleavage of Mismatch            *

Introduction     *

Reagents         *

Method            *

Microplate array diagonal gel electrophoresis (MADGE)    *

Introduction    *

The use of MADGE in simple PCR checking and SNP genotyping reactions   *

1: Standardization of PCR for subsequent MADGE analysis    *

2: MADGE principles and procedures  *

3: Preparation of MADGE gels *

4: Electrophoresis of MADGE gels        *

5: Visualization and Analysis of MADGE gels   *

6: ARMS and 192-well MADGE  *

Acknowledgements          *

Extraction of nucleic acids

Introduction

A number of different tissues are received in the laboratory for DNA extraction including whole blood, CVS samples, Guthrie blood spots, and wax block samples. Whole blood is always extracted by the adaptation of the Puregene method. The other sample types have different protocols which are described in the appropriate part of this manual.

General guidelines for all DNA extractions:

  1. A sample extraction sheet is prepared.
  2. Label all tubes to be used in the extraction procedure with the name of the patient and lab number.
  3. Keep the tubes in the work racks in numerical order.
  4. The names and lab numbers must be checked at all transfer stages throughout the extraction procedure to avoid sample mix up.
  5. Stagger the tubes in the rack to allow easy identification of samples which have been transferred.

DNA Extraction From Whole Blood

DNA Extraction Using A Variation Of The Puregene Method (Salt precipitation)

Nucleated cells are separated from whole blood by lysing the red cells followed by centrifugation to pellet the nuclei. Following white cell lysis contaminating proteins are removed by precipitating with a high concentration of ammonium acetate. DNA is then precipitated with isopropanol followed by washing the DNA with ethanol. The DNA is then dissolved in tris-EDTA and used for analysis.

Solutions

Red Blood Cell Lysis Solution (Autoclave)

155mM Ammonium Chloride                      8.3g per litre

10mM Potassium Bicarbonate       1g per litre

1mM EDTA                                        0.4g per litre OR 2 mls 0.5 M EDTA

White Cell Lysis Solution (Autoclave)

25mM EDTA                                     9.3g per litre OR 50 mls 0.5 M EDTA

2% SDS                                             20g per litre OR 200mls 10% SDS

Protein Precipitation Solution (Use Sterile Water, do not autoclave)

10M Ammonium Acetate                 385.4g per 500 mls

Ethanol

70% (v/v) ethanol, analar                  700mls per litre

Isopropanol

Isopropanol, analar

Glycogen (20mg/ml)

Glycogen                                            20mg per ml

N.B.     Store solutions at room temperature, except glycogen which is kept at

•           20oC.

            Do not use new solutions for important samples until they have been proven   reliable.

            RCL Buffer should be aliquoted in to 500 mls bottles as a reduction in yields have been noticed when approaching the end of a single large bottle.

Equipment

  1. MSE centrifuge with swing out rotor.
  2. 13mls polypropylene centrifuge tubes (Sarstedt LTD, 68 Boston Road, Beaumont Leys, Leicester LE4 1AW, Tel: 01533 359023).
  3. Plastic Pasteur dropping pipettes (Regional Supplies).
  4. Spiromix
  5. 37° C water bath.
  6. Fume cupboard.
  7. 2mls Microcentrifuge tubes (Sarstedt LTD, 68 Boston Road, Beaumont Leys, Leicester LE4 1AW, Tel: 01533 359023).
  8. Microcentrifuge.
  9. 1.5mls and 0.5mls Microcentrifuge tubes (Alpha Laboratories Ltd. 40 Parham Drive, Eastleigh, Hants. SO5 4NU)
  10. 0.5mls pipette tips (Alpha Laboratories Ltd. 40 Parham Drive, Eastleigh, Hants. SO5 4NU).
  11. 10mls pipette tips (Alpha Laboratories Ltd. 40 Parham Drive, Eastleigh, Hants. SO5 4NU).
  12. .Blue pipette tips (Alpha Laboratories Ltd. 40 Parham Drive, Eastleigh, Hants. SO5 4NU).
  13. Yellow pipette tips (Regional Supplies).
  14. Whirlymixer.
  15. Various sized automatic pipettes (Alpha Laboratories Ltd. 40 Parham Drive, Eastleigh, Hants. SO5 4NU).
  16. .Ice Maker
  17. Eye protection

Method

Whole Blood In EDTA Or Li/HEP

1. Check the order and alignment of the blood tubes and the tubes for transfer and to initial the extraction sheet to indicate this check has been made. Transfer 3 mls whole blood into a 15 mls polypropylene centrifuge tube.

2. Add 9 mls Red Cell Lysis solution (i.e. 3 volumes).

3. Spiromix for 10 minutes at room temperature.

4. Centrifuge at 3300 RPM (2000 X G) for 10 minutes in an MSE centrifuge fitted with swing-out rotor.

5. Pour off the Supernatant - this will leave behind ~200m l residual liquid.

6. Add 3 mls White Cell Lysis solution (i.e. 1 volume) to resuspended pellet - pipette up/down to lyse cells.

7. Invert several times - stable in this state at Room Temperature for 18 months.

8. Incubate for 30 minutes at 37° C.

9. Cool to below 21° C, 5 minutes on ice.

10. Add 1 mls Protein Precipitation solution.

11. Whirly mix for 20 seconds.

12. Centrifuge at 3300 RPM (2000 X G) for 10 minutes in an MSE centrifuge fitted with swing-out rotor.

13. Pour supernatant into a clean 15 mls polypropylene centrifuge tube.

14. Add 3 mls Isopropanol (propan-2-ol) and invert several times to precipitate DNA from the supernatant.

15. Centrifuge at 3300 RPM (2000 X G) for 10 minutes in an MSE centrifuge fitted with swing-out rotor.

16. Gently pour off the supernatant, blot onto a paper towel.

17. Add 3 mls 70% Ethanol to the DNA pellet, briefly whirly mix.

18. Centrifuge at 3300 RPM (2000 X G) for 10 minutes in an MSE centrifuge fitted with swing-out rotor.

19. Gently pour off the supernatant, blot onto a paper towel - ensure all liquid is removed.

20. Repeat 70% ethanol wash.

21. Check DNA pellet is at the bottom of the tube.

22. Leave in fume cupboard inverted for approximately 30 minutes to dry.

23. Resuspend in 110m l TE and leave at R.T. overnight.

24. Transfer to a DNA tube and measure the OD 260nm.

25. Adjust the volume of TE to give a standard concentration of 500/250 (m g/ml) if possible.

26. Leave on rotator for a few hours or overnight to mix.

Phenol-Chloroform Extraction

Nucleated cells are separated from whole blood by either lysing the red cells (using either ammonium chloride solution or mild detergent) followed by centrifugation to pellet the nuclei, or by ficoll gradient separation. These are then resuspended in buffered salt (STE) plus SDS and proteinase K to digest histones and other proteins. The DNA (and RNA) is then extracted in a two phase system using phenol and chloroform extraction. At pH 7 and above, DNA selectively partitions into the aqueous phase (although some is lost in the phenol phase). DNA is recovered by adding 2 volumes of ethanol followed by spooling onto a Pasteur pipette. The DNA is then dissolved in tris-EDTA and used for analysis. An alternative, more rapid method is to "salt out" (precipitate under conditions of high ionic strength) the proteins from STE solution and spool out the DNA without phenol extraction.

Solutions

1xSTE Plus 1% SDS (pH7.8) (Autoclave)

100mM NaCl                                     5.88g per litre

10mM tris (pH7.8)                             1.21g per litre

1mM EDTA                                        0.37g per litre OR 2 mls 0.5 M EDTA

1% SDS                                             10g per litre OR 100mls 10% SDS

Can be made by using: 100mls STE (10X) and 100mls 10% SDS up to 1 litre with water

TE Buffer ; pH8.0 (Autoclave)

10mM tris (pH8.0)                             0.121g per litre

1mM EDTA                                        0.037g per litre OR 0.22 mls 0.5 M EDTA

Can be made by using: 100mls TE (10X) upto 1 litre with water

Phenol Solution

Saturated solution in 0.1M tris from Northumbria Biologicals

Aliquot as 10 mls with a 0.1M tris overlay and store at -20° C

Chloroform

Chloroform, analar

Ethanol

Ethanol (100%), analar

Proteinase K

20mg/ml in water store at -80° c

Equipment

1. MSE centrifuge with swing out rotor.

2. 13mls (or 30mls) polypropylene centrifuge tubes (Sarstedt LTD, 68 Boston Road, Beaumont Leys, Leicester LE4 1AW, Tel: 01533 359023).

3. Plastic Pasteur pipettes (Regional Supplies).

4. Spiromix

5. 37° C water bath.

6. Fume cupboard.

7. 2mls Microcentrifuge tubes (Sarstedt LTD, 68 Boston Road, Beaumont Leys, Leicester LE4 1AW, Tel: 01533 359023).

8. Microcentrifuge.

9. 1.5mls and 0.5mls Microcentrifuge tubes (Alpha Laboratories Ltd. 40 Parham Drive, Eastleigh, Hants. SO5 4NU)

10.5mls pipette tips (Alpha Laboratories Ltd. 40 Parham Drive, Eastleigh, Hants. SO5 4NU)

11.Glass Pasteur pipettes (Merck Ltd., Hunter Boulevard, Magna Park, Lutterworth, Leics, LE17 4XN).

12.Blue pipette tips (Alpha Laboratories Ltd. 40 Parham Drive, Eastleigh, Hants. SO5 4NU).

13.Yellow pipette tips (Regional Supplies).

14.Whirlymixer.

15.Various sized automatic pipettes (Alpha Laboratories Ltd. 40 Parham Drive, Eastleigh, Hants. SO5 4NU).

16.Scalpels (Regional Supplies).

17.Weighing boats (Merck Ltd., Hunter Boulevard, Magna Park, Lutterworth, Leics, LE17 4XN).

Method

Lysis of fresh blood

If the blood is clotted then process only the unclotted part by this method (see "DNA extraction from clot or tissue if the sample is completely clotted). Centrifuge fresh blood at 3200rpm for 10 minutes to separate the cells. Red cells will be at the bottom, white cells (buffy coat) will be a thin layer overlaying this and plasma will be the top layer. Remove the upper layer to waste using plastic Pasteur. Remove the buffy coat ensuring that you take a portion of the red cells in-order to ensure all the buffy coat has been taken. Add 2.5 volumes of Red Cell Lysis buffer to the buffy coat and spiromix for 10 minutes. The solution will go from a murky red to a clear deep red/maroon colour. When blood clears, centrifuge at 3,200rpm for 10 minutes. Tip off the buffer. A fawn pellet of nuclei and white cells should be visible. It may be covered by some red cells, which can be gently washed off with red cell lysis buffer.

Lysis of frozen blood

Lyse whole blood (or a stored buffy coat) with 2.5 volumes of Red Cell Lysis buffer.

When blood clears, centrifuge at 3,200rpm for 10 min. Tip off the buffer. A fawn pellet of nuclei and white cells should be visible. It may be covered by some red cells, which can be gently washed off with red cell lysis buffer.

The pellet from whole blood lysates is generally smaller and more contaminated with red cell debris. These pellets should be cleaned up using a second lysis step with ice cold Red Cell Lysis buffer B, followed by centrifugation at 3,200 rpm once more. If the pellet is still discoloured do not use the salt extraction procedure, instead use phenol/chloroform extraction which will clean up the DNA better.

Digestion of white cells

1.         Add 5ml White Cell Lysis buffer (STE, SDS to 1%) and proteinase K to 50mg/ml. (50-100ml of 20mg/ml stock)

2. Mix gently. Solution will become very viscous.

3.         Incubate at 37oC overnight or 55oC for 2 hours.

 

Phenol/chloroform extraction

1.         Add 5ml phenol (use 10ml tubes) and mix gently.

2.         Centrifuge at 3,200 rpm for 15 minutes.

3.         Remove upper, aqueous phase using plastic Pasteur.

4.         If necessary, cut the end of the Pasteur to widen the bore for easier transfer of viscous solution.

5.         Remove aqueous phase and add 5ml of chloroform, repeat centrifugation as above.

6.         Remove aqueous phase and add 2-2.5 volumes of ethanol.

7.         Mix gently. The DNA will appear as a white fibrous precipitate which can be recovered by winding round a bent Glass Pasteur pipette.

8.         Remove the Pasteur and snap off the end into a 2ml Sarstedt tube, allow to dry in the fume cupboard.

9.         Add 110m l Tris-EDTA (TE) and dissolve by placing on a blood tube rotator for 1-3 days. The DNA can be dissolved faster by heating to 55oC if rapid analysis is needed.

 

Alternative - Salt extraction

  1. This protocol can be used in place of the phenol/chloroform extraction method.
  2. Add 0.5 volume of saturated NaCl or 7.5M NH4Ac to the STE) and shake vigorously for 20 seconds.
  3. Stand the tubes in ice/water for 15 minutes then centrifuge at 4,000 rpm for 20 minutes A pellet of denatured protein should form at the bottom of the tube.
  4. Gently remove the solution and transfer to a new 13ml tube.
  5. Add an equal volume of isopropanol (propan-2-ol) and spool out the precipitate.
  6. Dry off the DNA and dissolve in 110m l of TE buffer and dissolve by placing on a blood tube rotator for 1-3 days. The DNA can be dissolved faster by heating to 55oC if rapid analysis is needed.

DNA extraction from buccal samples (Qiagen system)

DNA extraction from Wax Blocks

When a patient is deceased the only source of material usually available from which DNA can be extracted is from a wax block. Wax blocks are booked into the lab system in the usual way and are stored in the cupboard labelled wax blocks in the extraction lab. If the whole block is received the method used to extract DNA usually results in damage to the block and as many pathology departments want the blocks back after we have used them permission to carry out the procedure must be obtained from the pathologist who sent the block or from the clinical geneticist involved.

Wax block material is not the best material for DNA extraction for a number of reasons:

1. The samples are usually old which increases the likelihood of DNA degradation.

2. The tissue samples have often been stored in formalin for extended periods prior to being embedded in wax which increases the likelihood of DNA degradation. As the samples are usually old it is difficult to find out exactly how the tissue was treated.

3. Degraded DNA is much more difficult to PCR and requires more amplification cycles.

4. DNA extracted from wax blocks can be contaminated with chemicals e.g formamide, wax, which may inhibit PCR completely.

5. The amount of DNA extracted is usually low and combined with degradation problems Southern blotting with wax block DNA is usually impossible.

Solutions

See Purgene Varients (Salt PPT.) extraction.

Equipment

See Purgene Varients (Salt PPT.) extraction.

Method

The method used is based on the Puregene extraction protocol.

1. Using a scalpel blade remove some of the tissue from the block and cut up in a clean weighing boat and transfer into a 15ml polypropylene tube. If shavings have been received transfer them straight into the polypropylene tube.

2. Add 2ml of white cell lysis solution and 20ml of proteinase K. Vortex to mix.

3. Incubate overnight at 55OC.

4. Check the digestion in the morning. If the solutio