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Figure 1.
Overview of REDExtract-N-Amp™ Tissue PCR Procedure.
Materials and Methods
All materials were obtained from Sigma-Aldrich (St. Louis, MO) unlessotherwise noted. All PCR primers were obtained from Sigma-Genosys(The Woodlands, TX).
Add Tissue Sample toTissue Preparation Solutionand Extraction SolutionIncubate for 10 minutesat room temperature.Heat 95
°
Cfor 3 min.Add Neutralization Solution.Mix aliquot withREDExtract-N-Amp™PCR ReadyMix™ and primers.Transfer to thermal cycler.PCRDirectly load amplified PCRproduct on gel.
Extract-N-Amp™ TissuePCR Kit: Rapid GenomicDNA Extraction fromTissue Coupled with PCR
By Scott Weber and Derek Douglas
Sigma-Aldrich Corporation, St. Louis, MO, USA
Introduction
Standard methods for extracting DNA from tissues can beextremely laborious and time consuming. Certain appli-cations, such as genotyping of transgenic mice using asection of tail, employ a lengthy DNA extraction process.For example, traditional mouse tail DNA extractions requirea 5 hour to overnight digestion with proteinase K andsubsequent phenol:chloroform extraction and alcoholprecipitation. Recently other options such as silica bindand elute kits have become available, but these alsorequire a 5 hour to overnight digestion of the tail beforethe DNA is extracted. These time consuming and laborintensive methods are necessary for some applications,but for more robust techniques, such as PCR, the DNAdoes not need to be isolated to the degree that thesemethods provide.PCR can amplify a target using very little template andfrom relatively impure starting material. Therefore, theExtract-N-Amp™ Tissue PCR Kit has been created to releasePCR-ready DNA from mouse tails in a 15 minute singletube procedure. The included 2x PCR mix is optimized towork with the crude extracts and the solutions in the kit.We describe a new method from which sufficient DNAfor PCR amplification is released from a mouse tail aftera 10 min extraction. The neutralized tissue extract is stablefor at least 6 months at 4 °C, and can be added directlyto the optimized PCR mix which is supplied in the kit.Furthermore, the Extract-N-Amp™ Tissue Kit works witha variety of source materials including fresh and frozenmouse tails, mouse tissue, buccal swabs, hair, saliva andsaliva archived on a collection card
.
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DNA extraction
Extraction Solution and Tissue Preparation Solution weremixed together and tissue samples were added to themixture (Figure 1). The mixture was incubated at roomtemperature for 10 minutes to extract the DNA. An incu-bation at 95 °C for 3 minutes stopped the extraction andNeutralization Solution B was then added to neutralize theextract. Samples were mixed thoroughly and used imme-diately in PCR. The volumes of the solutions used and incu-bation times for each starting material are listed inTable 1.*
PCR amplification
For standard PCR, 10
m
l of REDExtract-N-Amp™ PCRReadyMix was combined with forward and reverse primersat 0.4
m
M and 4
m
l of neutralized tissue extract in a finalvolume of 20
m
l. Reactions were assembled at roomtemperature. Cycling was performed in a GeneAmp
 ® 
PCR System 9700 thermal cycler (Perkin Elmer/AppliedBiosystems, Foster City, CA). The PCR primers used foreach sample type and the cycling parameters used arelisted inTable 1.*The sequences for the primers are pre- sented inTable 2.*Aliquots of 3-6
m
l of each PCR productwas loaded and fractionated on an ethidium bromide-stained 1% agarose gel in Figures 2 and 3.For quantitative PCR, 10
m
l of Extract-N-Amp™ PCRReadyMix was combined with forward and reverse primersat 0.4
m
M, SYBR
 ® 
Green I dye (Product Code S 9430),0.6
m
lof Reference Dye for Quantitative PCR (Product CodeR 4526),and 4
ml
of tissue extract in a final volume of20
ml
. Quantitative PCR was performed in an ABI PRISM
 ® 
7700 Sequence Detection System (Perkin Elmer/AppliedBiosystems, Foster City, CA). Primers and cycling parametersused are listed inTable 1.*Mouse DNA standards for quantitative PCR were pre-pared from 1.2 cm of mouse tail using the GenElute™Mammalian Genomic DNA Kit (Product Code G1N10, G1N70or G1N350).The purified DNA was diluted to 57.5 ng/ 
m
l and seven 2-fold serial dilutions were preparedfrom the initial dilution. Single-use aliquots of these DNAstandard dilutions were stored at –20 °C. A set of standardswas assayed along with the mouse tail extracts.
Sequencing
PCR products prepared from mouse-tail extracts with theIL-1
b
primer pair were sequenced after purification withthe GenElute™ PCR Clean-up Kit (Product Code NA1020). The sequence was obtained using BigDye™ TerminatorChemistry on an ABI PRISM
 ® 
377 DNA Sequencer(Applied Biosystems, Foster City, CA).
   O  r   d  e  r  :   1 .   8   0   0 .   3   2   5 .   3   0   1   0    T  e  c   h  n   i  c  a   l   S  e  r  v   i  c  e  :   1 .   8   0   0 .   3   2   5 .   5   8   3   2
Results and Discussion
PCR amplification from tissue extracts
The Extract-N-Amp™ Tissue PCR Kit was used to extractand amplify target DNA from mouse tails, mouse braintissue, buccal swabs, human hair, saliva and salivaarchived on a collection card
.
As shown in Figure 2, PCRproducts ranging from 1-2 kb were detected on ethidiumbromide stained 1% agarose gel from each extract. Thedata in Figure 2 demonstrates that PCR-ready DNA canbe rapidly extracted from a variety of tissue sources.Additionally, experiments are in progress using theExtract-N-Amp™ Tissue PCR Kit to extract DNA fromother starting materials such as
Drosophila
and fungi.
Figure 2. PCR analysis of genomic DNA extracted from various types of sources.
Extract-N-Amp™ Tissue PCR Kit was used to extract and amplify genomic DNA fromvarious sources. Genomic DNA was extracted using the protocol as described inMaterials and Methods. In all cases, the entire extraction procedure was completed in less then 15 minutes. All samples were then amplified using the specially formulated Extract-N-Amp PCR ReadyMix™ included in the kit. PCR products generated are1181 bp for the Interleukin-1
b
(IL-1
b
 ) gene in mouse and 1820 bp for the Carnitine palmitoyltransferase II (CPT-2) in human.
It is known that both the reaction conditions and thetemplate quality determine the size of PCR product thatcan be amplified.
1
Using the Extract-N-Amp™ Tissue PCRKit a 5 kb fragment of
b
-Globin (Figure 3) was successfullyamplified using extracts from buccal swab, hair, salivaand saliva archived on a collection card. The results shownin Figures 2 and 3 demonstrate that reaction conditionsand template quality obtained with the Extract-N-Amp™
M2 kb
 —
1.5 kb
 —
1 kb
 —
750 bp
 —
500 bp
 —
300 bp
 —
M
   M  o  u  s  e   T  a   i   l ,   I   L  -   1   M  o  u  s  e   B  r  a   i  n   T   i  s  s  u  e ,   I   L  -   1   B  u  c  c  a   l   S  w  a   b ,   C   P   T  -   2   H  a   i  r ,   C   P   T  -   2   S  a   l   i  v  a ,   C   P   T  -   2   S  a   l   i  v  a   C  a  r   d ,   C   P   T  -   2   N  e  g  a   t   i  v  e   C  o  n   t  r  o   l
 
AB
Figure 4. Quantitative PCR results showing mouse tail extract stability.
Four mousetails were extracted using the Extract-N-Amp Tissue PCR Kit following the proceduredescribed in Materials and Methods. Plot A shows the amplification of four mouse tail extracts immediately following extraction. Plot B shows the amplification of the stan-dards used to quantify the DNA in the mouse tail extracts. Reaction analyses were performed on an ABI Prism™ 7700
®
Sequence Detection System.
Half of the remaining extracts were stored at 4
°
C (recom-mended storage conditions) and the other half at 37
°
C(accelerated storage). Quantitative PCR was performedafter 5 and 10 weeks for all extracts and on separatealiquots of the same standards used at time zero. Asshown in Figure 5, mouse-tail extracts are stable at 37
°
Cfor 10 weeks. Results for extracts stored at 4
°
C are verysimilar to those shown in Figure 5 (data not shown). Itshould be noted that the mouse tail was removed fromthe neutralized extract before storage.
Figure 3. PCR analysis of genomic DNA extracted from various types of human tissueand amplified with 5 kb
b
-Globin primers.
Extract-N-Amp™ Tissue PCR Kit was used to extract and amplify genomic DNA from various human tissue sources. GenomicDNA was extracted from buccal swabs, hair, saliva, and saliva archived on a collectioncard using the protocol as described in Materials and Methods. All samples were thenamplified using the specially formulated Extract-N-Amp PCR ReadyMix™ included in thekit. The PCR products generated are 5 kb for a segment of the Human
b
-Globin gene.
Tissue PCR Kit are suitable for amplification of both small(< 2 kb) and medium (5 kb) sized PCR products.It should be noted that the Extract-N-Amp
™
ExtractionSolution contains components that are not compatiblewith standard PCR mixes. The 2x Extract-N-Amp
™
PCRMixes are specifically formulated to compensate forthese components and other impurities released duringthe extraction process. Extracts added to a PCR mixtureother than the Extract-N-Amp
™
or REDExtract-N-Amp
™
PCR ReadyMix will likely yield little or no PCR product.
Stability of mouse tail extracts
To test the stability of the DNA extracts prepared withthe Extract-N-Amp
™
Tissue PCR Kit, stability tests wereset up with mouse-tail extracts utilizing quantitative realtime PCR. Four mouse tails were extracted according to theprocedure outlined in the Materials and Methods sectionand 4-
m
l aliquots were analyzed immediately by quantita-tive PCR with SYBR
 ® 
Green dye detection on an ABI PRISM
 ® 
7700. The Extract-N-Amp
™
Tissue PCR Kit extracts areamplified without significant quenching of the SYBR Greensignal. As seen in Figure 4, the amplification plots for theextracts (A) fall in the middle of the amplification plotsfor the genomic mouse DNA standards (B).
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0246810121416182022242628303234363840A9B9E9F910^ 110^ 110^ 0A2B1C1D1E1024681012141618202224262830323436384010^ 110^ 0
5 kb
   1   k   b   l  a   d   d  e  r   B  u  c  c  a   l  s  w  a   b   H  a   i  r   S  a   l   i  v  a   N  e  g  a   t   i  v  e   C  o  n   t  r  o   l   1   k   b   l  a   d   d  e  r   S  a   l   i  v  a   f  r  o  m   a  c  o   l   l  e  c   t   i  o  n  c  a  r   d
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