Note: Descriptions are shown in the official language in which they were submitted.
`-'?~'I/03~ 1;37~8~ PCl/US93/07382
.
PROCESS FOR BTAINING A NON-LIQUID CELL SAMPLE
, ,
Back~ound of the Imention
Advances in recombinant DNA technology have made possible the
molecular analysis and prediction of several genetic disorders. At the present
time, ~enetic analyses are carried out mainly usin~ blood ieukocytes, which are
3 obtained from whole blood (e.g. via centrifu~ation). Whole blood, in turn must
be obtained clinically by a physician or phlebotomist employing an invasive
technique (e.g~ venipuncture).
i
A non-invasive method for obtaining genetic material using a mouthwash
has been describeci (Lench, N., et. al., The Lancet l: l356-8 (June l8, l988)). In
this method, an individual rinses his mouth with saline which is then emitted from
the mouth into o container, The container is centrifuged and cells, which are
pelleted upon centrifugatlon, are collected. The ease with which sampies can
be obtained is a major attraction of this method. However, samples so obtained
are in a liquid format and therefore present probiems with regard to handling,
2 o processing and contamination.
DNA for genetic analysis has also been obtained from cervicovaginal
cells ~Burk, R.D, ond C, Spitzer, Am. J. Obstet. GYnecol.l62:~52-4 (1990)). In astudy which compared the quantatlve yieid of cellular material obtained by
2 5 various methods, cervicovaginal lavage was found to be superior to scrape,
swab or brush methocls for obtaining cervicovaginal cells (Goldberg, (~L et. al.,
Am. J. Obstet. GYnecol~ 16l: l 169-72 (1989); V~rmund, SH et. ai., Am. J. Obstet.
Gvnecol. 160: 304-8 (1989)). However, cervicovaginal cells obtained by lavage
are in a liquid format and therefore present the same problems with handling,
; 1 ` ' 30 proaessing and contamination as scsmples obtained by mouthwashing
techniques.
~: '
j;
WO 94/031 1 1 ~ 1 3 7 2 ~ 2 P~/US93/0738 '~=
For forensic purposes. DNA has been o~taine~ from epithelial cells, spun
from urine (Gaspc~rinin, P. et. al., N. Enal. J. Med. 320: 80~ (1989)); and from root
and surrounding sheath cells obtained from plucked hairs (Higuchi, R. et. al.,
Nature 332:543-6 (1988)). However, samples so obtained often do not contain
S enough DNA for genetic analysis. Therelore hair and urine samples are not '
optimal for clinical ana~sis.
A simple, noninvasive technique for obtaining a non-liquid cell sample
having an appropriate amount of genetic material for clinical analysis ~ould be
1 0 useful.
Summar,r o~ the InvenHon
The invention relates to a simple, noninvasive process 'or obtaining a non-
liquid cell sample from an animal for genetic analysis. The process involves
inserting an appropriately srzed brush into an animal's oral cavily, contacting the
brush with an oral membrane, agr~ating the brush against the membrane so that
cells are captured within the bristles of the brush, removing the brush, which
contains the captured ce!ls and isolating the cells from the bristles of the brush for
use in genetic analysis. In a preferred embodiment, the brush is spiral, so thatcells can be obtained by rotating the brush against an oral mernbrane.
Cells so obtaineci can then be analyzed using any of a number of known
genetic techniques. For example, cells obtained according to the disclosed
method can be Iysed and the cell's DNA can be contacted with a suitable
labelled probe. In addition, DNA so obtained can first be amplified to increase
the amount of genetic material available for arlalysis.
A major advantage of using the process described herein for obtaining
~ I cells for genetic testing is that the non-liquid sample so obtained is easier to
handle and to process than a liquid sample. Further a non-liquid sample is less
prane to contamination (e.g by bacteria~ and is more stable.
,
J--`-'94~03ll~ 21~7X82 Pcr/US93/0738'`
l` ` '.
Detailed ~e~ ot the Invention
In general, the invention relates to a process for obtainlng a non-liquid cell
sample from an animal (e.g. a human or a domesticated mammal) for genetic
analysis. The process employs a brush to obtain cells from within a animal's oral
cavity. Exarr ples of cells which can be obtained from an animal's oral cavity
include epithelial cells of the inner cheek or tongue.
Accordin~ to the method of the invention, a brush is inserted into an
anlmal's oral cavity, contacted with an oral membrane and agitated (e.g.
rotated or brushed) against the membrane for a sufficient period of time, so that
cells are captured within the bristles of the brush. The brush. which contains
captured cells is then removed from the animal's mouth and cells are isolated
from the bristles of the brush for use in genetic analysis.
To be suitable for use in the subject invention, a brush must be of an
appropriate size to enable insertion into the animal's oral cavity. In addition, the
brush bristles should be free of potentially contaminating material. For example,
a brush comprising mammalian hair should not be used in the subject method,
2 0 because genetic material present within the hair could contaminate the sample
and thereby interfere with the genetic analysis. Preferred brushes for use in the
subJect invention have synthetic (e.g. nylon or docron) bristles. An especially
preferr~d brush for use in ths invention is the nylon bristle cytology brush,
(CytosottTM Medical Packaging Corporation, Camarillo, CA), which is currently
2 5 used for Pap Smear and Chlamydia testing.
Once removed from the mouth, a brush which contains captured oral
cells can be analyzed directly or transported andlor stored for subsequent
genetic analysis. At thls point, measures aimed at avoiding the dislodging of
~ I captured cells from a brush should be taken. For example, the brush can be
pl~ced in a container (e.g. an envelope) during transportation and/or storage.
Cells may t~ obtained from a brush by any method which avoids loss of
captured cells. A preferred method of obtaining cells from th t brush is by
:
,:
,.~ ,
~ ~ .
:`
WO 94/031 1 1 PCr/US93/0738'~
2137282 '~
contacting the cells with a solution. For example, if DNA is to be isolated from the
cells for amplification, the cells c~n be obtained from the brush using a solution
which Iyses cells (e.~. sodium hydroxide, potassium hydroxide).
Genetic material from oral celis so obtained can then be analyzed
genetically using any of a number of known genetic techniques, such as
di~estion with restriction endonucleases, ultraviolet light visualization of ethidium
i~romld~ stalned a~arose g81s, DNA sequencing, or hybridization with detectable
nucloic acid sequences ~e.~, labelled probes). In addi~ion, the genetic materialfrom cells obtalned accordins to the disclosed method can be amplified using
known arnplification techniques, such as Q-beta-replicase and the polymerase
chain reaction (PCR) (Saiki, R.K. et. al~, Science 239:487-491 (1~88); Wong, C., et.
al., Nature 330, 384-386 ~1987~ and U.S. Patent 4,683,202 to Mullis et. al.) to further
increase the amount of genetic material available for genetic analysis.
The invention will now be illustrateci in the following Example, which is not
intended to be limiting in any way.
Example 1: Obtaining Cells From Oral Cavities Using either a Brush or a Wooden
2 0 Spatula
The oral cavities of five human subJects were scraped with wooden
spatulas for about 30 seconds, and in five human subjects, the CytosottTM nylon
bristle cytology brushes (Medical Packa~ing Corporation; Camarilo, CA) were
2 5 rotatad against the inner side of their cheeks for about 30 seconds. The spatulas
or brushes were soaked in test tubes containing 50mM NaOH for about 1 hour,
a~ter whlch one tenth volume of 1 M TRIS (pH8) was added to the test tubes to
neutralke.
3 0 , I The cheok c811 Iysates obtained using the spatulas or the brushes were
used directiy in à po!ymerase chain reaction ~PCR) to amplify one or mofe of théfollowing exons of the Cystic Fibrosis transmembrane regulator gene: exons 4, 10,
11, 20 anci 21. Apropriats primers for the PCR were deYeloped based on the
nuclelc acid sequence cilsclosed in Riordan, J.R. et al., Science 245:1066-1073
~1989).
~`-` ') 94/03]i 1 1 PCI`/US93/07382
. l `` 2 t~282
PCR amplification reactions were performed using AmpliTaq DNA
polymerase and a DNA Thermal Cycler (Perkin Elmer). Cheek Iysates were jl
amplified for 28 cy~les of 1û seconcis at 94, 10 seconds 5~C, and 30 seconds at
74C, with a final soak for 10 minutes at 74C. The reaction buffer used was
described previousiy (Saiki, R.K. et ai., Science 239:487-491 {1988)~.
Ampiified materiai was then blotted onto nyion membranes (Biotrans Plus,
iCN i3iomedical) and PCR products were analyzed on 1% agarose gels. No PCR
products were detected for any of the five samples in which cells were obtained
usin~ a wooden spatula. In contrast, PCR products were detected for all five
samples obtained using a brush. These PCR products were hybridized to
sequence specific probes to identify known mutations (Kerem. B. et al., Science
245:1073-1080 (1989); Lemna et al., N. Ena. J. Med. 322:291-296 (1990); Kerern et
ai., Proc. Natl. Acad. Sci. USA 87:8447-8451 (1990).
Equivalents:
Those skilled in the art will recognize or be ab!e to ascertain using no more
than routine experimentation, many equivalents to the specific embodiments of
2 0 the invention dascribed specificaiiy herein. Such equivalents are intended to be
encompassed In the scope of the foilowing cic~ims.
t'.
~: .
, ~
S
; : q
: - 5 -
` :~
~ ~,
