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INTRODUCTION
TECHNICAL FIELD
The field of this invention is fluorescent tags and their use as
exemplified with DNA fragment analysis.
BACKGROUND
There is an increasing demand to be able to identify and quantify
components of mixtures. The greater the complexity of the mixture, the
greater the interest in being able to simultaneously detect a plurality of
the components present. As illustrative of this situation is DNA
sequencing and DNA fragment analysis, where it is desirable to efficiently
excite from one to four or more fluorescently tagged components with a
laser source at a single wavelength, while providing for fluorescent
signal emission at a plurality of distinctive wavelengths. In this
situation, the different labels should not adversely affect the
electrophoretic mobility of DNA fragments to which they are attached.
Currently, there are four methods used for automated DNA sequencing: (1)
the DNA fragments are labeled with one fluorophore and then the fragments
run in adjacent sequencing lanes (Ansorge et al., Nucleic Acids Res. 15,
4593-4602 (1987); (2) the DNA fragments are labeled with four different
fluorophores and all the fragments are electrophoretically separated and
detected in a single lane (Smith et al., Nature 321, 674-679 (1986); (3)
each of the dideoxynucleosides in the termination reaction is labeled with
a different fluorophore and the four sets of fragments are run in the same
lane (Prober et al., Science 238, 336-341 (1987); or (4) the sets of DNA
fragments are labeled with two different fluorophores and the DNA
sequences coded with the dye ratios (Huang et al., Anal. Chem. 64,
2149-2154 (1992). For fluorescence based PCR DNA fragments analysis,
primers labeled with different fluorescent dyes were employed thereby
permitting multiple target analysis (Andy et al. (1995) Biotechniques, 18,
116-121).
All of these techniques have significant deficiencies. Method 1 has the
potential problems of lane-to-lane variations in mobility, as well as a
low throughput. Methods 2, 3 and 4 as well as the multiple color PCR
method require that the four dyes be well excited by one laser source and
that they have distinctly different emission spectra. In practice, it is
very difficult to find two or more dyes that can be efficiently excited
with a single laser and that emit well separated fluorescent signals.
As one selects dyes with distinctive red-shifted emission spectra, their
absorption maxima will also move to the red and all the dyes can no longer
be efficiently excited by the same laser source. Also, as more different
dyes are selected, it becomes more difficult to select all the dyes such
that they cause the same mobility shift of the labeled molecules.
It is therefore of substantial interest that improved methods be provided
which allow for multiplexing of samples, so that a plurality of components
can be determined in the same system and in a single run. It is also
desirable for each label to have strong absorption at a common wavelength,
to have a high quantum yield for fluorescence, to have a large Stokes
shift of the emission, that the various emissions be distinctive, and that
the labels introduce the same mobility shift. It is difficult to
accomplish these conflicting goals by simply labeling the molecules with a
single dye.
SUMMARY OF THE INVENTION
The subject invention provides compositions and methods for analyzing a
mixture using a plurality of fluorescent labels. To generate the labels,
pairs or families of fluorophores are bound to a backbone, particularly a
nucleic acid backbone, where one of the members of the families is excited
at about the same wavelength. By exploiting the phenomenon of energy
transfer, the other members of each of the families emit at detectably
different wavelengths. The range of distances between donor and acceptor
chromophores is chosen to ensure efficient energy transfer. Furthermore,
labels used conjointly are selected to have approximately the same
mobility shift in a separation system, where one of the labels may have
two molecules of the same fluorescer, so as to provide the fluorescence
emission of the single fluorescer, but the same mobility shift as the
different donor-acceptor chromophore labels. This is achieved by changing
the mobility shift of the labeled entity, by varying the distance between
the two or more members of the family of fluorophores and by choosing
labels with the same mobility. The subject invention finds particular
application in DNA sequencing and DNA fragment sizing, where the
fluorophores may be attached to universal or other primers and different
fluorophore combinations used for the different dideoxynucleosides. Kits
of combinations of labels are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in color.
Copies of this patent with color drawings will be provided by the Patent
and Trademark Office upon request and payment of the necessary fee.
SCHEME 1. Structures of the four energy transfer (ET) primers (SEQ ID NOs
1-4) and a representative synthetic scheme for the preparation (SEQ ID NOs
5 and 6) of F6R. The fluorescent primers are labeled with a common
fluorescein donor (F) at the 5' end and either a second fluorescein or a
rhodamine (R) acceptor at the indicated locations of a modified T in the
sequence. The number of nucleotides between the two fluorophores is
indicated in the primer designation.
FIG. 1 shows the absorption (----) and fluorescence emission (.sub.------)
spectra of the fluorescently labeled THO1 primers F14F and F6R measured in
1.times.TBE. F14F exhibits strong absorption at .about.488 nm and intense
fluorescence emission with a maximum at 525 nm. F6R also exhibits intense
absorption at .about.488 nm but the maximum emission is shifted out to
.about.600 nm.
FIG. 2 shows the capillary electrophoresis (CE) electropherogram of primer
F6R and ROX labeled primer. The two primers with the same sequence and
same molar concentration were mixed together in 80% formamide and analyzed
with a polyacrylamide gel filled capillary. The fluorescence signals were
detected in the green (----) and red (.sub.------) channels simultaneously
with 488 nm excitation (argon laser). The fluorescence signal intensity of
F6R is 8-fold higher than that of ROX labeled primer.
FIG. 3 shows the capillary electrophoresis electropherograms of (A) the
THO1 standard ladder consisting of the 6, 7, 8, and 9 alleles which have
been mixed and then coinjected with a .phi.X-174 RF DNA-HincII digest. (B)
Standard allelic ladder spiked with "unknown" allele 7. (C) Standard
allelic ladder spiked with allele 9. (D) Standard allelic ladder spiked
with alleles 7 and 8. (E) Standard allelic ladder spiked with alleles 9
and 9.3. These separations were run with 0.8% hydroxyethyl cellulose
(HEC), 1/2.times.TBE and 1 .mu.M thiazole orange in the running buffer and
detected in the green channel. The additional weak peaks appearing at the
detection times greater than .about.17 min are due to heteroduplex
formation.
FIG. 4 shows the comparison of the mobility shift using five different
methods for attaching energy-transfer coupled fluorophores to THO1 target
alleles 6 and 9.3. The green line indicates the fluorescence intensity in
the green channel and the red line indicates the intensity in the red
channel. The structures of the primer sets used are indicated.
Electrophoresis was performed using 0.8% HEC, 1/2.times.TBE and 1 .mu.M
9-aminoacridine (9-AA) in the running buffer.
FIG. 5 shows the images of the fluorescence from a five capillary array
separation of THO1 alleles. The left image presents the fluorescence
signal as a function of time detected in the red (>590 nm) channel while
the right image presents the fluorescence signal from the green
(.lambda.max=525 nm) channel. The standard THO1 allelic ladder (6+7+8+9)
was amplified with the red emitting ET primer F6R and detected in the red
channel; unknown alleles were amplified with the green emitting primer
F14F and detected in the green channel. These images have been adjusted
for the 1-2% capillary-to-capillary variance in mobility by shifting the
time axes so that the allelic ladder is detected at the same time in all
capillaries. This separation was performed with 0.8% HEC and 1 .mu.M 9-AA
in the running buffer at 80V/cm.
FIG. 6 shows the electropherograms of the THO1 fragment sizing separations
presented in FIG. 5. The green signal is from the unknown alleles and the
red signal is from the standard THO1 ladder. Traces A through E correspond
to lanes 1-5 in FIG. 5.
FIG. 7 shows the CE electropherogram of single strand (ss) DNA fragments
generated with F10F and F3R primer and with ddATP/dNTPs. Green and red
traces correspond to F10F and F3R DNA fragments which were detected in the
green (.lambda.max=525 nm) and red (.lambda.>590 nm) channels
respectively. ssDNA fragments were generated using Sequenase sequencing
kit (USB/Amersham LIFE SCIENCE).
DESCRIPTION OF SPECIFIC EMBODIMENTS
Novel fluorescent labels, combinations of fluorescent labels, and their use
in separation systems involving the separation of a plurality of
components are provided. Particularly, the fluorescent labels comprise
pairs of fluorophores, which with one exception where the fluorophores are
the same, involve different fluorophores having overlapping spectra, where
the donor emission overlaps the acceptor absorption, so that there is
energy transfer from the excited fluorophore to the other member of the
pair. It is not essential that the excited fluorophore actually fluoresce,
it being sufficient that the excited fluorophore be able to efficiently
absorb the excitation energy and efficiently transfer it to the emitting
fluorophore.
The donor fluorophores in the different families of fluorophores may be the
same or different, but will be able to be excited efficiently by a single
light source of narrow bandwidth, particularly a laser source. The donor
fluorophores will have significant absorption, usually at least about 10%,
preferably at least about 20% of the absorption maxima within 20 nm of
each other, usually within 10 nm, more usually within 5 nm, of each other.
The emitting or accepting fluorophores will be selected to be able to
receive the energy from donor fluorophores and emit light, which will be
distinctive and detectably different. Therefore, one will be able to
distinguish between the components of the mixture to which the different
labels have been bound. Usually the labels will emit at emission maxima
separated by at least 10 nm, preferably at least 15 nm, and more
preferably at least 20 nm.
Usually the donor fluorophores will absorb in the range of about 350-800
nm, more usually in the range of about 350-600 nm or 500-750 nm, while the
acceptor fluorophores will emit light in the range of about 450-1000 nm,
usually in the range of about 450-800 nm. As will be discussed
subsequently, one may have more than a pair of absorbing molecules, so
that one may have 3 or more molecules, where energy is transferred from
one molecule to the next at higher wavelengths, to greatly increase the
difference in wavelength between absorption and observed emission.
The two fluorophores will be joined by a backbone or chain, usually a
polymeric chain, where the distance between the two fluorophores may be
varied. The physics behind the design of the labels is that the transfer
of the optical excitation from the donor to the acceptor depends on
1/R.sup.6, where R is the distance between the two fluorophores. Thus, the
distance must be chosen to provide efficient energy transfer from the
donor to the acceptor through the well-known Foerster mechanism. Thus, the
distance between the two fluorophores as determined by the number of atoms
in the chain separating the two fluorophores can be varied in accordance
with the nature of the chain. Various chains or backbones may be employed,
such as nucleic acids, both DNA and RNA, modified nucleic acids, e.g.
where oxygens may be substituted by sulfur, carbon, or nitrogen,
phosphates substituted by sulfate or carboxylate, etc., polypeptides,
polysaccharides, various groups which may be added stepwise, such as
di-functional groups, e.g. haloamines, or the like. Of particular interest
is nucleic acid, particularly DNA and RNA as the backbone, where the bases
may be naturally occurring or synthetic, particularly naturally occurring.
The fluorophores may be substituted as appropriate by appropriate
functionalization of the various building blocks, where the fluorophore
may be present on the building block during the formation of the label, or
may be added subsequently, as appropriate. Various conventional
chemistries may be employed to ensure that the appropriate spacing between
the two fluorophores is obtained. In the case of the label having two
molecules of the same fluorophore, the spacing will be selected to have
the same mobility as a label having two molecules having different
fluorophores. The label with the same fluorophores need not have efficient
energy exchange, so that there is substantial flexibility in the
separation of the same fluorophores to achieve the desired mobility.
However, the two fluorophores should be spaced to avoid self quenching, so
that the two fluorophores will usually be spaced more than about 2
nucleotides apart.
The molecular weights of the labels (fluorophores plus the backbone to
which they are linked) will generally be at least about 250 Dal and not
more than about 20,000 Dal, usually not more than about 10,000 Dal. The
molecular weight of the fluorophore will generally be in the range of
about 250 to 1,000 Dal, where the molecular weights of the acceptor-donor
pairs on different labels to be used together will usually not differ by
more than about 20%. The fluorophores may be bound internal to the chain,
at the termini, or one at one terminus and another at an internal site.
The fluorophores may be selected so as to be from a similar chemical
family, such as cyanine dyes, xanthenes or the like. Thus, one could have
the donors from the same chemical family, each donor-acceptor pair from
the same chemical family or each acceptor from the same family or the
cross combination of the family.
The subject labels find particular application in various separation
techniques, such as electrophoresis, chromatography, or the like, where
one wishes to have optimized spectroscopic properties, high sensitivity
and comparable influence of the labels on the migratory aptitude or
mobility of the components being analyzed. Of particular interest is
electrophoresis, such as gel, capillary, etc. Among chromatographic
techniques are HPLC, affinity chromatography, thin layer chromatography,
paper chromatography, and the like.
It is found that the spacing between the two fluorophores will affect the
mobility of the label. Therefore, one can use combinations of the same dye
pair and different dye pairs and by varying the distance between the dye
pairs, within a range which still permits good energy transfer for the
different dye pairs, provide for substantially constant mobility for the
labels. The mobility is generally not linearly related to the specific
spacing, so that one will empirically determine the effect of the spacing
on the mobility of a particular label. However, because of the flexibility
in the spacing of the fluorophores in the labels, by synthesizing a few
different labels with different spacings and different dye pairs, one can
now provide for a family of fluorescent labels, which share a common
excitation, that have strong and distinctive emission and a substantially
common mobility. Usually, the mobility will differ by not more than about
20% of each other, preferably not more than about 10% of each other, and
more preferably within about 5% of each other, when used in a particular
separation. The mobility may usually be determined by carrying out the
separation of the labels by themselves or the labels bound to a common
molecule which is relevant to the particular separation, e.g. a nucleic
acid molecule of the appropriate size, where one is interested in
sequencing or sizing.
A wide variety of fluorescent dyes may find application. These dyes will
fall into various classes, where combinations of dyes may be used within
the same class or between different classes. Included among the classes
are dyes, such as the xanthene dyes, e.g. fluoresceins and rhodamines,
coumarins, e.g. umbelliferone, benzimide dyes, e.g. Hoechst 33258,
phenanthridine dyes, e.g. Texas Red, and ethidium dyes, acridine dyes,
cyanine dyes, such as thiazole orange, thiazole blue, Cy 5, and Cyfr,
carbazole dyes, phenoxazine dyes, porphyrin dyes, quinoline dyes, or the
like. Thus, the dyes may absorb in the ultraviolet, visible or infra-red
ranges. For the most part, the fluorescent molecules will have a molecular
weight of less than about 2 kDal, generally less than about 1.5 kDal.
The energy donor should have a strong molar absorbance coefficient at the
desired excitation wavelength, desirably greater than about 10.sup.4,
preferably greater than about 10.sup.5 cm.sup.-1 M.sup.-1. The absorption
maximum of the donor and the emission maximum of the acceptor (fluorescer)
will be separated by at least 15 nm or greater. The spectral overlap
integral between the emission spectrum of the donor chromophore and the
absorption spectrum of the acceptor chromophore and the distance between
the chromophores will be such that the efficiency of energy transfer from
donor to acceptor will range from 20% to 100%. Separation of the donor and
acceptor based on number of atoms in the chain will vary depending on the
nature of the backbone, whether rigid or flexible, involving ring
structures or non-cyclic structures or the like. Generally the number of
atoms in the chain (the atoms in the ring structures will be counted as
the lowest number of atoms around one side of the ring for inclusion in
the chain) will be below about 200, usually below about 150 atoms,
preferably below about 100, where the nature of the backbone will
influence the efficiency of energy transfer between donor and acceptor.
While for the most part, pairs of fluorophores will be used, there can be
situations where up to four different, usually not more than three
different, fluorophores bound to the same backbone may find use. By using
more fluorophores, one may greatly extend the Stokes shift, so that one
may excite in the visible wavelength range and emit in the infra-red
wavelength range, usually below about 1000 nm, more usually below about
900 nm. Detecting light in the infra-red wavelength range has many
advantages, since it will not be subject to interference from Raman and
Rayleigh light resulting from the excitation light. In order to maintain
the mobility constant, one may use the same number of fluorophores on the
labels, having a multiplicity of the same fluorophore to match the number
of fluorophores on labels having different fluorophores for the large
Stokes shift.
The subject invention finds particular application with nucleic acid
chains, where the nucleic acid chains find use as primers in sequencing,
the polymerase chain reaction, particularly for sizing, or other system
where primers are employed for nucleic acid extension or ligation and one
wishes to distinguish between various components of the mixture as related
to the particular labels. For example, in sequencing, universal primers
may be employed, where a different pair of fluorophores are used for each
of the different dideoxynucleosides used for the extension during
sequencing.
A large number of nucleosides are available, which are functionalized, and
may be used in the synthesis of a polynucleotide. By synthesizing the
subject nucleic acid labels, one can define the specific sites at which
the fluorophores are present. Commercially available synthesizers may be
employed in accordance with conventional ways, so that any sequence can be
achieved, with the pair of fluorophores having the appropriate spacing.
Where different primers have been used in PCR, each of the primers may be
labeled in accordance with the subject invention, so that one can readily
detect the presence of the target sequence complementary to each of the
different primers. Other applications which may find use include
identifying isozymes, using specific antibodies, identifying lectins using
different polysaccharides, and the like.
Also, of great interest is the use of the subject labels with rapid sizing
of alleles, as exemplified by short tandem repeat (STR) alleles, or other
sequences where one wishes to detect small base or base pair differences,
such as small differences of as few as a single base or base pair. By
using the subject labels in conjunction with capillary electrophoresis,
particularly capillary array electrophoresis, and employing an
intercalating agent in the buffer, separations differing by one base may
be achieved. The method can be used with dsDNA, particularly dsDNA
obtained using the polymerase chain reaction or the ligase chain reaction,
where the subject labels may be used as primers. One or both of the
primers for the amplification may be labels, where the fluorophore pairs
may be the same or different, depending on the needs of the separation.
The intercalating agents may be fluorescent or non-fluorescent, such as
thiazole orange, 9-aminoacridine, ethidium bromide, and the like, but for
the specific example give here they are preferably non-fluorescent.
Concentrations will generally be in the range of 0.1 to 10 .mu.M.
Conventional conditions may be used for the capillary electrophoresis,
using a polyacrylamide wall coating and, for example, using
hydroxyethylcellulose at from about 0.5 to 1% in an appropriate running
buffer. Voltages may vary from about 50 to 150V/cm or larger. The amount
of DNA will generally be in the range of about 1 pg/.mu.l to 1 ng/.mu.l,
although greater or lesser amounts may be used. Obviously, such method can
also be used for single strand (ss) DNA fragment analysis to detect the
labelled ssDNA fragments by virtue of their fluorescence, using linear
polyacrylamide or the like in CE, which permits single base resolution.
Kits are provided having combinations of labels, usually at least 2. Each
of the labels will have the acceptor-donor pair, usually with comparable
backbones, where the labels will be separated along the backbone to give
comparable mobility in the separation method to be used. Each of the
labels in a group to be used together will absorb at about the same
wavelength and emit at different wavelengths, where one or more of the
labels may conveniently have the same fluorescer as the donor and the
acceptor. In each kit there will usually be at least one label with
different fluorescers as the donor and acceptor. In order to be able to
use the same excitation source, the labels having the same fluorescer for
the pair, should have substantially overlapping absorption spectra, and
different emission spectra. Therefore, the number of different labels
capable of being used in a set, where the individual label has a common
chromophore will be relatively limited. Each of the labels in the group
will have about the same effect on mobility in the separation method, as a
result of the variation in placement of the different fluorophores along
the backbone.
The kits will generally have up to about 6, usually about up to about 4
different labels which are matching in mobility, but may have 2 or more
sets of matching labels, having 2-6 different labels.
Of particular interest are labels comprising a nucleic acid backbone, where
the labels will generally have at least about 10 nucleotides and not more
than about 50 nucleotides, usually not more than about 30 nucleotides. The
labels may be present on the nucleotides which hybridize to the
complementary sequence or may be separated from those nucleotides. The
fluorophores will usually be joined to the nucleotide by a convenient
linking arm of from about 2 to 20, usually 4 to 16 atoms in the chain. The
chain may have a plurality of functionalities, particularly
non-oxo-carbonyl, more particularly ester and amide, amino, oxy, and the
like. The chain may be aliphatic, alicyclic, aromatic, heterocyclic, or
combinations thereof, usually comprising carbon, nitrogen, oxygen, sulfur,
or the like in the chain. The fluorophores will usually be separated by
not more than about 20 nucleotides, usually not more than about 15
nucleotides.
The entire nucleic acid sequence may be complementary to the 5' primer
sequence or may be complementary only to the 3' portion of the sequence or
to any portion within the target sequence. Usually, there will be at least
about 4 nucleotides, more usually at least about 5 nucleotides which are
complementary to the sequence to be copied. For PCR reactions the primers
are combined with the sequence to be amplified along with Taq polymerase
and dNTPs. After amplification, the DNA may be isolated and transferred to
a gel or capillary for separation.
The kits which are employed will have at least two of the subject labels,
which will be matched by having substantially the same absorption for the
donor molecule, distinct emission spectra and substantially the same
mobility. Generally for single stranded nucleic acids, the separation will
be from about 2-20, more usually 2-15, preferably about 2-14 nucleosides
between fluorophores.
The following examples are offered by way of illustration and not by way of
limitation.
Rapid Sizing of Short Tandem Repeat (STR) Alleles Using Energy-Transfer
(ET) Fluorescent Primers and Capillary Array Electrophoresis
Experimental Instrumentation. Capillary array electrophoresis separations
were detected with the laser-excited, confocal-fluorescence scanner as
previously described by Huang et al. (Huang et al. (1992) Anal. Chem.
1992, 64, 967-972. and Anal. Chem. 1992, 64, 2149-2154). Briefly,
excitation light at 488 nm from an argon ion laser is reflected by a
long-pass dichroic beam splitter, passed through a 32.times., N.A. 0.4
microscope objective, and brought to a 10 .mu.m diameter focus within the
75 .mu.m i.d. capillaries in the capillary array. The fluorescence is
collected by the objective, passed back through the first beam splitter to
a second dichroic beam splitter that separates the red (.lambda.>565 nm)
and green (.lambda.<565 nm) detection channels. The emission is then
focused on 400 .mu.m diameter confocal pinholes, spectrally filtered by a
590 nm long-pass filter (red channel) or a 20 nm band-pass filter centered
at 520 nm (green channel), followed by photomultiplier detection. The
output is preamplified, filtered, digitized, and then stored in an IBM
PS/2 computer. A computer-controlled stage is used to translate the
capillary array past the optical system at 20 mm/s. The fluorescence is
sampled unidirectionally at 1500 Hz/channel. The scanner construction and
operation have recently been described in detail (Mathies et al. (1994),
Rev. Sci. Instrum. 65, 807-812). Postacquisition image processing was
performed with the programs IPLab, KaleidaGraph and Canvas.
Capillary Electrophoresis. Polyacrylamide-coated, fused-silica capillaries
were prepared using a modification of the procedure described by Hjerten
et al. ((1985), J. Chromatogr. 347, 191-198). A 2-3 mm wide detection
window was produced by burning off the polyimide coating with a hot wire
followed by cleaning the external surface with ethanol. The detection
window was placed 25 cm from the injection ends of the 75 .mu.m i.d., 350
.mu.m o.d., 50 cm long fused silica capillaries (Polymicro Technologies,
Phoenix, Ariz.). The inner walls of the capillaries were incubated with 1N
NaOH for 30 min at room temperature, followed by rinsing with deionized
water. The capillaries were then treated overnight at room temperature
with .gamma.-methacryloxypropyl-trimethoxysilane (1:250 dilution with
H.sub.2 O adjusted to pH 3.5 with acetic acid) to derivatize the walls for
acrylamide binding. Freshly-made 4% T acrylamide solution in 1/2.times.TBE
buffer (45 mM tris, 45 mM boric acid, 1 mM EDTA, pH 8.3) was filtered with
a 0.2 .mu.m syringe filter and degassed under vacuum for 30 min. One .mu.l
TEMED (tetramethylethylenediamine) and 10 .mu.l of 10% APS (ammonium
persulfate) solution were added to 1 ml of gel solution. The solution was
immediately forced into the capillary with a 100-.mu.l syringe. After 30
min, the acrylamide solution was flushed out with deionized water and
capillaries were filled with buffer consisting of hydroxyethyl cellulose
(HEC) (M.sub.n =438,000, Aqualon Co. Hopewell, Va.) dissolved in
1/2.times.TBE. The separation buffer was prepared by adding 0.8 g HEC to
100 ml 1/2.times.TBE and dissolved by stirring overnight at room
temperature. The HEC buffer was degassed under vacuum for 30 min,
centrifuged for 20 min on a tabletop centrifuge, drawn into a 100-.mu.l
syringe, and 3 .mu.l sample was used for injection into each capillary.
Capillaries were prerun at 80 V/cm for 5 min before each experiment.
Diluted and deionized PCR samples were injected by inserting the capillary
in a 5-.mu.l sample volume held in an Eppendorf tube followed by
electrokinetic injection (80 V/cm for 3 s). After injection, the sample
tubes were replaced with tubes containing 0.8% HEC plus 1/2.times.TBE
buffer. Electrophoresis was performed at 80 V/cm using 5-capillary arrays
held at ambient temperature (22.degree. C.). The low (80 V/cm)
electrophoresis voltage was used to avoid undersampling of the bands with
our current detection system which is limited to 1 Hz scan rates. When the
experiments were complete, capillaries were flushed with water, then with
methanol followed by drying. These coated capillaries could be refilled
20-25 times before the quality of the separations deteriorated. Methods
for the further extension of the lifetime of capillary columns have been
described.
PCR Amplification of THO1 loci. DNA was isolated from blood by using
standard methods (Puers et al. (1993) Am. J. Hum. Genet. 53, 953-958). The
human tyrosine hydroxylase locus HUMTHO1, chromosomal location 11p15.5,
contains a polymorphic four base STR sequence (AATG) in intron 1 ›Puers,
1993, 953!. PCR-amplification of this polymorphic region produces allelic
fragments designated "5" through "11", according to the number of AATG
repeats; an additional allele designated "9.3" differs from allele 10 by a
single base deletion. The primer sequences used for PCR are
5'-ATTCAAAGGGTATCTGGGCTCTGG-3' (THO1-A) SEQ ID NO:6 and
5'-GTGGGCTGAAAAGCTCCCGATTAT-3' (THO1-B) SEQ ID NO:7 (Edwards et al. (1991)
Am. J. Hum. Genet. 49, 746-756). PCR amplifications were performed in 50
.mu.l volumes by using 10 ng genomic DNA template, 0.5 .mu.M of each
primer, 5 units Taq DNA polymerase, 50 mM KCl, 1.5 mM M.sub.g Cl.sub.2, 10
mM Tris-HCl at pH 8.3, and 200 .mu.M dNTPs (final concentrations
indicated). The PCR cycle protocol using a Perkin Elmer Cetus Model 480
was: (1) melting at 95.degree. C. for 5 min, (2) 30 cycles of 95.degree.
C. for 1 min, 58.degree. C. for 1 min, and 72.degree. C. for 1 min, (3)
72.degree. C. for 7 min to complete extension. The PCR sample was then
dialyzed for 30 min by pipeting 8-10 .mu.l onto a 0.10 .mu.m VCWP membrane
filter (Millipore, Bedford, Mass.) which was floated on de | | |
