MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
PRINCIPLE:
Chelex® 100 (Chelex) is a chelating resin that has a high affinity for polyvalent metal ions. The
Chelex resin is composed of styrene divinylbenzene copolymers containing paired iminodiacetate
ions that act as chelating groups. It is postulated that the presence of Chelex during boiling
prevents the degradation of DNA by chelating metal ions that may otherwise catalyze the
breakdown of DNA subjected to high temperatures in low ionic strength solutions or prevent
metal ions from activating nucleases. The basic procedure consists of a wash to remove some of
the contaminants such as heme and proteins, followed by boiling the sample in a 5% Chelex
solution. A fraction of the supernatant is added to the PCR reaction.
SPECIMEN:
Any biological sample believed to contain nucleated cells.
REAGENTS AND SPECIAL SUPPLIES:
Bleach, 10% (7 mM sodium hypochlorite solution)
Centricon-100 microconcentrator or Microcon-100 microconcentrator
Chelex® 100, 5% or 20% suspension, (Bio-Rad)
Dithiothreitol (DTT), 1 M
Forceps
Milli-Q Type I water
pH Indicator Strips
Proteinase K (20 mg/ml)
Scalpel, disposable
Sperm Wash Buffer
Spin-Ease microcentrifuge tubes (for sperm extractions)
Tips, aerosol-resistant (e.g., for P-10, P-100, P-1000 pipettes)
Tris-EDTA (TE-4), (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA)
Tubes, (1.7 ml, or 15 ml)
Weigh boats
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
INSTRUMENTATION AND EQUIPMENT:
Boiling Water Bath
Freezer, -20oC
Dry Block Incubators (37oC, 56oC)
Magnetic Stir bars
Magnetic Stirring Plate
Microcentrifuge (e.g., Eppendorf)
Pipettes (e.g., P-10, P-100, P-1000)
Refrigerator, 4oC
Vortex
FRESH REAGENT PREPARATION:
5% and 20% suspensions of Chelex® 100 must be made fresh weekly using Milli-Q water. pH
the suspension daily before use. The pH must be between 9.0 - 10.0. Do not attempt to adjust
the pH.
QUALITY ASSURANCE:
1.
Extractions of specimens are to be performed in the extraction laboratory only.
2.
Ensure that the following worksheet is completed for this procedure and placed in the
appropriate case folder: "Chelex Extraction of DNA.”
3.
Ensure that all reagents satisfy the minimum standards for quality control.
4.
Ensure that all equipment satisfies the minimum standards for quality control, where
appropriate.
5.
In order to prevent contamination, all steps in this procedure should be conducted in the
proper laboratory hoods when appropriate.
6.
Any laboratory workspace and all pipettes and racks to be used in this procedure must be
cleaned with 10% bleach and thoroughly dried before beginning. When using a laminar
flow hood, turn on the ultraviolet light for a minimum of 10 minutes after cleaning.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
7.
Before use, forceps, scissors, etc., must be cleaned with 10% bleach, rinsed in ethanol, and
dried thoroughly. In addition, they should be cleaned in the same manner following the
processing of each individual specimen (when used).
8.
To prevent contamination, microcentrifuge tubes must be irradiated in an ultraviolet
crosslinker with 2 j/cm2. The extraction buffer and TE buffer (in 50 ml conical tubes) must
be irradiated with 6 j/cm2. Follow "Irradiation of Reagents and Supplies in the Ultraviolet
Crosslinker" SOP for appropriate method of irradiation.
9.
No reagents or supplies used for the extraction of DNA from specimens will be allowed in
a post-amplification room.
10.
Any equipment taken from a post-amplification room to a pre-amplification room must be
sterilized with 10% bleach immediately before removal from the post- amplification room
and immediately upon arrival in the pre-amplification room. Reagents should never be
transferred from a post-amplification room to a pre-amplification room.
11.
Only one evidence specimen will be open at any one time.
12.
Change pipette tips between each transfer or addition of sample or reagent, unless
otherwise noted.
13.
No aliquot of any reagent may be returned to the original stock container.
14.
A minimum of one reagent blank must be carried throughout the extraction procedure and
assayed in parallel with the evidence samples.
15.
Extraction Reagent Blank Control: an extraction reagent blank control must be initiated for
each set of extraction tubes. The volume of reagent blank amplified must be no less than
the volume of evidentiary sample extracted. This control will regulate for the presence of
contamination in the extraction reagents. The reagent blank should be the first tube setup
during purification. The extraction reagent blank must be carried through the remaining
analyses.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
SAFETY:
1.
All appropriate MSDS sheets must be read prior to performing this procedure.
2.
Treat all biological specimens as potentially infectious. Gloves, safety glasses, and a
laboratory coat must be worn at all times. Follow Universal Precautions.
3.
Avoid direct exposure to ultraviolet light when using the germicidal lamp in the biological
hood or the transilluminator.
4.
The heat plate can become very hot. Be careful not to touch the heating surfaces while in
operation.
5.
Use proper protective equipment to prevent burns when handling boiling water or hot
solutions.
6.
Distinguish all waste as general, biohazard, or SharpsTM and discard appropriately.
PROCEDURAL NOTES:
1.
Wipe down work area with 10% bleach. Use gloves and aerosol pipette tips to minimize
contamination. Change or clean gloves and instruments between each sample.
2.
When pipetting Chelex solutions, the resin beads must be distributed evenly in solution.
This can be done by gentle mixing with a stir bar in a beaker. Use a pipette tip with a large
bore (P-1000 µl).
3.
DNA extracted with Chelex is single-stranded and should be quantitated by slot blot since
it is unsuitable for quantitation methods that use ethidium bromide.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION FROM WHOLE BLOOD AND BLOODSTAINS
FOR BLOODSTAINS:
1. Take cutting of swab or fabric (may be cut into small pieces) and place in a labeled tube.
NOTE: The sample size will be dependent upon the quantity of material available and
whether the sample appears to be heavily stained or not. Generally, take approximately a
1/8” diameter punch or a ½ cm2 if the stain is very heavy, and up to approximately 1 ½ cm2 if
the stain is light.
2. Add 1 ml of TE-4 to the tube.
FOR LIQUID BLOOD:
1. Add 1 ml of TE-4 into a labeled tube.
2. Add 3-5 l of well-mixed whole blood.
3. Mix and incubate at room temperature for 15-30 minutes.
4. Spin for 2-3 minutes at maximum speed (10,000 - 15,000 rpm).
5. Remove and discard all but approximately 50 l of the supernatant. Leave substrate and
pelleted material in the tube.
OPTION: The supernatant can be used to verify the presence of human hemoglobin using the
HemaTrace test if the stain is thought to be less than approximately 1 ½ years old. See “One
Step ABAcard Hematrace” Procedure.
6. Resuspend the pellet in 200l or more of 5% Chelex to submerge the entire substrate.
7. Incubate at 56o C for a minimum of 1 hour (for fresh whole blood and blood stains), or up to
24 hours if the stain is resistant to hydration.
8. Vortex for 5 - 10 seconds.
9. Incubate in a boiling water bath for 8 minutes.
10. Vortex for 5 - 10 seconds.
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CHELEX® 100 EXTRACTION OF DNA
EXTRACTION FROM WHOLE BLOOD AND BLOODSTAINS (CONTINUED)
11. Spin for 2-3 minutes at maximum speed.
OPTION: DNA extracts may be concentrated using “Concentration of DNA with Microcon-100
and Centricon-100” protocol.
12. Store at 2-8o C for short-term storage. Freeze at -20o C (or colder) for long-term storage. To
use after long-term storage on Chelex beads, repeat steps 8 through 11.
NOTE: If the color of the extracted sample is a dark red brown color, the potential for inhibition
must be considered.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION FROM SEMEN-CONTAINING STAINS
Starting from cutting or swab:
1.
Dissect swab or fabric into small pieces and place in a labeled tube.
NOTE: The sample size will be dependent upon the quantity of material available, whether the
sample appears to be heavily stained or not, and whether the material is lightweight or
heavyweight. Generally, use ½ of a swab if there is only one swab, or use an entire swab head if
there is more than one swab; for light-weight fabric or gauze, use approximately 1 ½ cm2
cuttings; for heavy-weight fabric (such as jeans or upholstery), use as much material as can easily
fit in a tube and leave sufficient room for mechanically agitating the substrate.
2.
Add 800 l of TE-4 to the tube. Vortex for at least 10 seconds (and/or agitate with a sterile
pipette tip) to aid in releasing cells from the substrate.
3.
Incubate at room temperature for 30 minutes, or longer at 4o C.
4.
Vortex for at least 10 seconds (and/or agitate with a sterile pipette tip) again.
5.
Transfer the substrate with a sterile pipette tip or forceps into a Spin-Ease Basket and place
the basket in the top of the corresponding tube.
6.
Spin for 5 minutes at maximum speed to elute the liquid and cells from the substrate in the
basket.
7.
Remove and discard the Spin-Ease Basket and substrate.
8.
Without disturbing the pellet, remove all but approximately 50 l of the supernatant (or
leave behind twice the volume of the pellet, whichever is greater) and save the supernatant
in a new tube for PSA testing in case no sperm are observed.
9.
Resuspend the cell debris pellet (potentially containing both sperm and epithelial cells)
thoroughly in the remaining supernatant (by pipetting up and down vigorously), and then
remove an aliquot equal to one-tenth of the volume in the tube for quantification of the
sperm/epithelial cell content. Follow SOP “Microscopic Examination of Cell Debris.”
OPTION: The supernatant can be used to verify the presence of semen using the OneStep
ABAcard p30 Test. (See “OneStep ABAcard PSA Determination Procedure”.)
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION FROM SEMEN-CONTAINING STAINS (CONTINUED)
Starting from PSA extraction cell pellet:
10. Add 150 l Milli-Q water to the approximately 50 l of cell debris pellet (final volume
should be approximately 200 l).
11. Add 2 l proteinase-K (20 mg/ml). Vortex briefly.
12. Incubate at 37oC for approximately 1 hour to lyse epithelial cells, but for no more than 2
hours to minimize the lysis of sperm cells.
13. Spin for 5 minutes at maximum speed. This original tube is now labeled with an “S”, “Sp”,
or “sperm” and contains the resulting sperm pellet.
14. Without disturbing the sperm pellet, transfer all but approximately 50 l of the supernatant
(or leave behind twice the volume of the pellet, whichever is greater) to a new tube, labeled
with an “E”, “Ep”, or “Epi” and contains epithelial cell DNA.
15. Add 50 l of 20% Chelex solution to the epithelial tube. Save at room temperature or 4oC
until step 22 below.
16. Wash sperm pellet (S) with Sperm Wash Buffer as follows: Resuspend the pellet in 500 l
Sperm Wash Buffer. Vortex briefly. Spin for 5 minutes at maximum speed. Remove and
discard all but 50 l (or twice the volume of the pellet, whichever is greater) of the
supernatant, being careful not to disturb the pellet.
17. Repeat step 16 an additional 2-3 times, for a maximum of 4 washes.
18. Wash sperm pellet (S) once with Milli-Q water as follows: Resuspend the pellet in 1 ml
water. Vortex briefly. Spin for 5 minutes at maximum speed. Remove and discard all but
50 l (or twice the volume of the pellet, whichever is greater) of the supernatant.
OPTION: Resuspend the pellet and remove a one-tenth aliquot for a microscopic examination to
assess the quantity of sperm present and the cleanliness of the extract. Follow SOP “Microscopic
Examination of Cell Debris.”
19. Add 150 l of 5% Chelex to the sperm pellet (final volume of approximately 200 l).
20. Add 2 l of proteinase-K (20 mg/ml) and 7 l of 1 M DTT. Mix gently.
21. Incubate at 37oC for 30 - 60 minutes.
22. Vortex epithelial (Ep) and sperm (S) cell samples for 5-10 seconds.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION FROM SEMEN-CONTAINING STAINS (CONTINUED)
23. Incubate in a boiling water bath for 8 minutes.
24. Vortex for 5-10 seconds.
25. Spin for 3 minutes at maximum speed.
OPTION: DNA extracts may be concentrated using “Concentration of DNA with Microcon-100
and Centricon-100” protocol.
26. Store at 2-8o C for short-term storage. Freeze at -20o C (or colder) for long-term storage. To
use after long-term storage on Chelex beads, repeat steps 22 through 25.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION OF DNA FROM TISSUE
1. Using a clean cutting surface for each different sample, dissect a piece of tissue
approximately 1 mm square (or larger) and place in a labeled microcentrifuge tube
2. Add 200l or more of 5% Chelex to submerge the entire substrate.
3. Incubate at 56o C for 30 minutes.
4. Vortex for 5-10 seconds.
5. Incubate in a boiling water bath for 8 minutes.
6. Vortex for 5-10 seconds.
7. Spin for 3 minutes at maximum speed.
OPTION: DNA extracts may be concentrated using “Concentration of DNA with Microcon100 and Centricon-100” protocol.
8. Store at 2-8o C for short-term storage. Freeze at -20o C (or colder) for long-term storage.
To use after long-term storage on Chelex beads, repeat steps 4 through 7.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION OF DNA FROM BONE
1. Weigh and measure bone sample before extracting. Photodocumentation is optional.
2. Using a clean surface for each different sample, scrape and sand the surface of the bone to
remove debris and potential foreign contamination using dremel tool with new, UV-irradiated
sanding disks.
3. Cut a section of bone using dremel tool with new, UV-irradiated cutting blades.
4. Weigh and measure the bone section to be analyzed.
5. Grind the bone sample in a bleached, alcohol-wiped, Waring blender.
6. Transfer the bone dust to a 15 ml conical tube.
7. Add sufficient 5% Chelex to submerge the entire substrate.
8. Incubate at 56o C for 30 minutes.
9. Vortex for 5-10 seconds.
10. Incubate in a boiling water bath for 8 minutes.
11. Vortex for 5-10 seconds.
12. Spin for 3 minutes at maximum speed.
OPTION: DNA extracts may be concentrated using “Concentration of DNA with Microcon-100
and Centricon-100” protocol.
13. Store at 2-8o C for short-term storage. Freeze at -20o C (or colder) for long-term storage. To
use after long-term storage on Chelex beads, repeat steps 9 through 12.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION OF DNA FROM SALIVA ON FABRIC OR SWABS
1. Dissect swab or fabric into small pieces and place in a labeled tube. If sample is too large for
a microcentrifuge tube, use a 15 ml conical tube.
NOTE: The sample size will be dependent upon the quantity of material available and whether
the sample appears to be heavily stained or not. Generally, use ½ of a swab if there is only one
swab, or use an entire swab head if there is more than one swab; for stained fabric or gauze, use
approximately 1 ½ cm2 cuttings.
2. Add 200l or more of 5% Chelex to submerge the entire substrate.
3. Incubate at 56oC for 30 minutes.
4. Vortex for 5-10 seconds.
5. Incubate in a boiling water bath for 8 minutes. When using 15 ml conical tubes, unscrew top
approximately ½ turn to allow tubes to vent pressure.
6. Vortex for 5-10 seconds.
7. Spin for 3 minutes at maximum speed.
OPTION: DNA extracts may be concentrated using “Concentration of DNA with Microcon-100
and Centricon-100” protocol.
8. Store at 2-8o C for short-term storage. Freeze at -20o C (or colder) for long-term storage. To
use after long-term storage on Chelex beads, repeat steps 4 through 7.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION OF DNA FROM ENVELOPE FLAPS AND STAMPS
1.
Use steam to carefully open the envelope flap or remove the stamp from envelope.
2.
Wet a cotton-tipped swab with Milli-Q water and swab adhesive area of the flap or stamp
with two cotton tipped swabs to remove any epithelial cells that may be present. Save one of
the two swabs for potential retesting. Alternatively, swab only half of the adhesive area,
leaving the second half undisturbed for potential retesting.
3.
Cut the swab head off and place into a labeled tube.
4.
Add 200l or more of 5% Chelex to submerge the entire substrate.
5.
Vortex a minimum of 10 seconds.
6.
Incubate at 56oC for approximately 30 minutes.
7.
Vortex for 5-10 seconds.
8.
Incubate in a boiling water bath for 8 minutes.
9.
Vortex for 5-10 seconds.
10. Spin for 3 minutes at maximum speed.
OPTION: DNA extracts may be concentrated using “Concentration of DNA with Microcon-100
and Centricon-100” protocol.
11. Store at 2-8o C for short-term storage. Freeze at -20o C (or colder) for long-term storage. To
use after long-term storage on Chelex beads, repeat steps 7 through 10.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION OF DNA FROM CIGARETTE BUTTS
1. Slice a strip approximately 5 mm wide from the paper covering the cigarette butt filter in the
area that would have been in contact with the mouth.
2. Remove the paper slice from the cigarette butt, cut it into smaller pieces, and put the pieces
into a sterile tube.
3. Add 200 l or more of 5% Chelex to submerge the entire substrate.
4. Incubate at 56oC for 30 minutes.
5. Vortex for 5-10 seconds.
6. Incubate in a boiling water bath for 8 minutes.
7. Vortex for 5-10 seconds.
8. Spin for 3-5 minutes at maximum speed.
9.
Concentrate DNA extracts using “Concentration of DNA with Microcon-100 and Centricon100” protocol.
10. Store at 2-8o C for short-term storage. Freeze at -20o C (or colder) for long-term storage. To
use after long-term storage on Chelex beads, repeat steps 5 through 8.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
EXTRACTION OF DNA FROM HAIR
1. Handling hair with clean forceps, examine hair under a microscope for the presence of sheath
material.
2. Use a clean scalpel to cut approximately 0.5 cm from the root end of the hair and place in a
labeled microcentrifuge tube. If the hair has not been washed for microscopic analysis,
immerse the hair root cutting in Milli-Q water to reduce surface dirt and contaminants, then
vortex at high speed for 10 seconds. Decant or pipet the water out of the tube. Repeat if
necessary.
3. Add 200 l or more of 5% Chelex to submerge the cuttings.
4. Add 5 l of proteinase-K (20 mg/ml).
5. Incubate at 56oC for at least 6 to 8 hours or overnight, making sure the hairs are completely
submerged in the Chelex solution.
6. Vortex for 5-10 seconds.
7. Incubate in a boiling water bath for 8 minutes.
NOTE: Check that the hair is completely immersed in the Chelex solution before boiling.
8. Vortex for 5-10 seconds.
9. Spin for 3 minutes at maximum speed.
11. Concentrate DNA extracts using “Concentration of DNA with Microcon-100 and Centricon100” protocol.
12. Store at 2-8o C for short-term storage. Freeze at -20o C (or colder) for long-term storage. To
use after long-term storage on Chelex beads, repeat steps 7 through 10.
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MAINE STATE POLICE CRIME LABORATORY: FORENSIC BIOLOGY SECTION
CHELEX® 100 EXTRACTION OF DNA
REFERENCES:
AmpliType User Guide, Version 2, Perkin Elmer Corporation, 1992.
Walsh, P. Sean, Metzger, David A. and Higuchi, Russell, "Chelex 100 as a Medium for Simple
Extraction of DNA for PCR-Based Typing from Forensic Material", Biotechniques Vol. 10 (4),
1991.
Reviewed
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Supervisor, Forensic DNA Section
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Director, Crime Laboratory
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