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sdfsdf

Paula F. Campos and Thomas M. P. Gilbert Abstract

The principal challenges facing PCR-based analyses of DNA extracted from formalin-fi xed materials are fragmentation of the DNA and crosslinked protein–DNA complexes. Here, we present an effi cient protocol to extract DNA from formalin-fi xed or paraffi n-embedded tissues (FFPE). In this protocol, protein–DNA crosslinks are reversed using heat and alkali treatment, yielding signifi cantly longer fragments and larger amounts of PCR-amplifi able DNA than standard DNA extraction protocols.

Key words:
Formalin , Formalin-fi xed , Paraffi n-embedded , DNA extraction , Ancient DNA 1. Introduction

The quality of DNA present in formalin-fi xed material is generally poor, due both to fragmentation undergone by the DNA molecule itself, in particular when unbuffered formalin is used as a fi xative, and to the formaldehyde-driven crosslinking of DNA with pro-

teins ( 1– 7
) . As a result, genetic analyses of such material must restrict themselves to targeting relatively short fragments of potentially amplifi able DNA. Ultimately, the quality of the DNA in a formalin-fi xed sample is diffi cult to predict, as the degree of degradation and crosslinking results from a complex interplay of factors including the duration, pH, strength, and temperature of the fi xative, plus the amount of time that has passed and the conditions of storage since fi xation. As a general rule, long fi xation times, in particular if longer than 12–24 h, highly acidic fi xatives, longer periods of time in storage, and storage in warm environments all contribute to DNA decay. Over time, these factors will lead to the destruction of any remaining DNA in the sample.

A large number of methods have been proposed to recover

DNA from formalin-fi xed material

(
for a review see r
ef.

( 8 )
)
.

Given that it is currently diffi cult, if not impossible, to reverse the Beth Shapiro and Michael Hofreiter (eds.),
Ancient DNA: Methods and Protocols
, Methods in Molecular Biology, vol. 840, DOI 10.1007/978-1-61779-516-9_11, © Springer Science+Business Media, LLC 2012

81

82

P.F. Campos and T.M.P. Gilbert

effects of DNA fragmentation, in our opinion, the key steps of successful methods are those that act through breakage of the DNA–protein crosslinks using either heat or alkali treatment, or both. In this chapter, we report a modifi cation of a method
( 9, 10
) that we have found previously to be both simple and extremely effective in this regard. The method involves an initial incubation of the fi xed tissue in a hot alkali buffer, coupled with a subsequent organic purifi cation of the nucleic acids. The method results in similar total yields of DNA per sample (as measured in m g/ m L) compared to other extraction methods such as commercial kits marketed with the specifi c aim of processing formalin-fi xed or paraffi

n-embedded (FFPE) material. However, the method

increases both the length of PCR-amplifi able fragments and the quantity of long-fragment DNA, as measured through conventional and quantitative real-time PCR
( 8– 10
) .

2. Materials

Prepare the digestion buffer using molecular biology grade reagents at room temperature, using appropriate anti-contamination controls (e.g., fi lter-tipped pipettes, DNA-free consumables, etc.). To minimize the risk of contamination, we recommend purchasing ready-made solutions, as opposed to making them in the laboratory.

1. Alkali digestion buffer: 0.1 M NaOH with 1% SDS solution.

Store at room temperature. The pH should be around 12.

2. 25:24:1 phenol:chloroform:isoamyl alcohol.

3. Chloroform.

4. Isopropanol.

5. 3 M sodium acetate, approximate pH 5.

6. (
Optional
) DNA precipitation “carrier,” e.g., Glycoblue (Ambion, Inc., Austin, TX).

7. Molecular Biology Grade ethanol, 85%.

8. TE elution buffer: 10 mM Tris–HCl, 1 mM EDTA (pH 8.0).

9. Microtome with sterile (DNA-free) blades or sterile scalpel blades.

10. Sterile 2-mL O-ring screw-top tubes.

11. Sterile 1.5-mL centrifuge tubes.

12. Centrifuge(s) for 1.5/2-mL tubes (>10,000 ×
g
).

13. Autoclave capable of heating to 120°C (preferable).

Alternatively, water bath or hotblock heated to 100°C.

14. Tabletop vortex.

11 DNA Extraction from Formalin-Fixed Material

83

3. Methods

Carry out all procedures at room temperature unless otherwise specifi ed. Always incorporate extraction blanks into the analysis.

This protocol is suitable for either paraffi n-embedded or non-embedded, formalin-fi xed material (see Note 1).

3.1. Tissue

1. Obtain small subsamples of tissue. For paraffi n-embedded tis-

Pre-Preparation

sue, use a microtome to obtain several slices of tissue between 3 and 10 m m thick, or shave thin slices using a sterile scalpel blade (see Note 1). For non-paraffi n-embedded tissue, obtain thin slices with a sterile scalpel blade.

3.2. Tissue Digestion

1. Place tissue in 0.5 mL of the alkali digestion buffer in a 2-mL

screw-cap O-ring tube (see Notes 2 and 3).

2. Use an autoclave to heat the tissue-buffer to 120°C for 25 min.

If autoclave use is not convenient, heating on a heat block or in a boiling water bath at 100°C for 40 min is an alternative (see Note 4).

3. Allow the tissue-buffer to cool for 5 min to room temperature (see Note 5).

4. Add 500 m L 25:24:1 phenol:chloroform:isoamyl alcohol to the mixture (see Note 6).

5. Agitate gently at room temperature for 5 min.

6. Centrifuge for 5 min at >10,000 ×
g
to separate the layers.

7. Carefully remove the upper aqueous layer and add to a new tube containing 500 m L chloroform. Be careful not to remove the protein-containing interface. Discard the lower phenol layer (see Note 6).

8.
Repeat steps 5–6 in Subheading 3.2
.

9. Remove the upper aqueous layer and place in a new 1.5 mL

eppendorf tube. Discard the lower chloroform layer (see

Note 6).

10. Add 0.6–1 volume isopropanol and 0.1 volume 3 M sodium acetate (approx. pH 5). A small amount of commercial carrier solutions can also be added if required to facilitate pellet visualization, such as Glycoblue (Ambion, Inc., Austin, TX), following the manufacturers’ guidelines. Mix well (see Note 7).

11. Immediately centrifuge at high speed (>10,000 ×
g
) for 30 min at room temperature.

12. Immediately following centrifugation, decant the liquid from the tube carefully. The DNA will have precipitated into a pellet at the bottom of the tube and may not be visible.

84

P.F. Campos and T.M.P. Gilbert

13. To rinse the pellet, gently add 500–1,000 m L 85% ethanol, gently invert once, then centrifuge for 5 min at high speed.

14. Gently decant the ethanol. Repeat if necessary.

15. All ethanol must be removed from the pellet as any residual ethanol will inhibit downstream applications. This can be easily achieved with a small bore pipette, followed by a brief incubation at a relatively high temperature (e.g., 55–75°C).

16. Resuspend the pellet in a suitable volume of TE buffer or ddH O (e.g., 50–100 m L). If the pellet has become very dry, 2

leave it at room temperature in the liquid for 5–10 min, followed by gentle pipetting.

4. Notes

1. It is not necessary to use a solvent to remove paraffi n from paraffi n-embedded samples prior to extraction; however, if present in large amounts, it is helpful to trim it away with a sterile scalpel fi rst.

2. The use of O-ring screw-cap tubes is extremely important, as under subsequent heating, high pressure will build up in the tube. Lids on tubes without an O-ring seal and screw fi tting will be blown open.

3. Once added, the alkali digestion solution will begin to degrade the DNA, thus delays in the subsequent steps (up to precipitation) should be avoided.

4. It takes time to heat up and cool down an autoclave, thus 25 min should represent the time at 120°C and not the entire time in the autoclave. The use of cooler temperatures (100°C) is not as effective as the original protocol, but nevertheless yields signifi cant improvements over other methods that do not incorporate a heat step.

5. The tissue will not have fully dissolved. This does not affect the results.

6. Organic extractions use phenol and chloroform to help purify the DNA. Both phenol and chloroform are toxic, and phenol

in particular is extremely dangerous. Neither should be used without appropriate local training. Always handle both liquids and their containers with extreme care, using appropriate face, hand, and body protection. Do not handle using latex gloves as these are permeable to phenol and chloroform; use only

nitryl gloves. The fumes of both are dangerous, thus always manipulate in a vented fume hood. Disposal of both requires conformation to specifi c regulations, thus relevant local disposal regulations should be consulted.

11 DNA Extraction from Formalin-Fixed Material

85

7. Isopropanol precipitation is most effective at relatively high centrifugal forces and in small tubes with a pointed end (the area covered by the precipitated DNA that forms the observed DNA pellet is most concentrated and thus easiest to spot and resuspend if 1.5-mL tubes or smaller are used).

Acknowledgments

MTPG was supported by the Danish National Science Foundation’s “Skou” grant program.