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Claims  |
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We claim:
1. A method for mapping a DNA segment made up of duplex strands of
nucleotides by
cleaving the segment with a first restriction enzyme to produce fragments
of DNA, each having a cleaved end,
attaching a reporter specific to a cleaved end nucleotide in each fragment,
cleaving the DNA fragments with a second restriction enzyme to produce
short fragments,
separating the short fragments according to size, and
analyzing the short, separated fragments for the presence of reporters, the
size and reporter identity being indicative of the character of each DNA
fragment.
2. The method as set forth in claim 1 wherein the segment cleaving produces
fragments of DNA with each cleaved end having one strand with a residue of
nucleotides and a corresponding recessed strand and includes the step of
attaching a reporter labeled nucleotide to the recessed strand to form a
base pair with at least one residue nucleotide, the reporter being
specific to each type of nucleotide.
3. The method as set forth in claim 2 wherein a third enzyme is used in
conjunction with and in addition to the first enzyme or second enzyme to
cleave different length fragments.
4. The method as set forth in claim 1 wherein a third enzyme is used in
conjunction with and in addition to the first enzyme or second enzyme to
cleave different length DNA fragments.
5. The method as set forth in claim 3 wherein the analysis is accomplished
in real time.
6. The method of claim 2 wherein the segment cleaving step produces DNA
fragments having a 5' overhang residue of nucleotides.
7. The method of claim 2 wherein the segment cleaving step produces DNA
fragments having a 3' overhang residue of nucleotides.
8. The method of claim 7 wherein the reporter attachment is accomplished by
using a polymerase exhibiting 3' exonuclease activity.
9. The method of claim 8 wherein the polymerase is selected from the group
consisting of DNA polymerase I, T7 DNA polymerase, and T4 DNA polymerase.
10. The method of claim 2 wherein the segment cleaving step produces DNA
fragments having blunt ends.
11. A method of mapping DNA segments made up of duplex strands of
nucleotides using a reporter comprising:
reacting a mixture of DNA segments and a first restriction enzyme that
cleaves the DNA to provide DNA fragments each with 5' or 3' overhang
strands and a corresponding 3' or 5' recessed strand or with blunt ends,
incubating the DNA fragments with a DNA polymerase and a mixture of
unlabeled deoxynucleotides and specific reporter labeled
dideoxynucleotides to add nucleotides to the recessed strands
complementary to the nucleotides in the overhang strands,
reacting the incubated DNA fragments with a second restriction enzyme to
produce short DNA fragments some of which include the reporter labeled
dideoxynucleotides and hence are reporter labeled,
separating the short DNA fragments according to size, and
detecting a reporter for each labeled fragment thereby identifying the 3'
terminal added dideoxynucleotides.
12. The method as set forth in claim 11 which includes the step of:
correlating the size and reporter identification of each labeled short DNA
fragment of the DNA segment.
13. The method as set forth in claim 12 wherein the ratio of labeled
dideoxynucleotides to unlabeled deoxynucleotides is varied inversely
according to the number of nucleotides in the overhang strands.
14. The method as set forth in claim 11 wherein a third enzyme is used in
conjunction with and in addition to the first enzyme or second enzyme to
cleave the DNA segments into different length fragments.
15. The method as set forth in claim 11 wherein the DNA fragments are
reacted only with reporter labeled dideoxynucleotides.
16. The method as set forth in claim 11 wherein the first restriction
enzyme is an ambiguous-end restriction enzyme.
17. The method as set forth in claim 13 wherein the first restriction
enzyme is an ambiguous-end restriction enzyme.
18. The method set forth in claim 11 wherein the first restriction enzyme
is an exact-end restriction enzyme.
19. The method set forth in claim 13 wherein the first restriction enzyme
is an exact-end restriction enzyme.
20. The method as set forth in claim 11 wherein the ratio of labeled
dideoxynucleotides to unlabeled deoxynucleotides is varied inversely
according to the number of nucleotides in the overhang strands.
21. The method as set forth in claim 13 wherein a third enzyme is used in
conjunction with and in addition to the first enzyme or second enzyme to
cleave the DNA segments into different length fragments.
22. The method as set forth in claim 13 wherein the DNA fragments are
reacted only with reporter labeled dideoxynucleotides.
23. The method as set forth in claim 13 wherein the first restriction
enzyme is an ambiguous-end restriction enzyme.
24. A method for mapping DNA segments made up of duplex strands of
nucleotides using specific binding pairs, one member of the pair being
immobilized on a solid support, by the steps of:
(a) cleaving each segment with a first restriction enzyme to produce
fragments of DNA each having a cleaved end,
(b) derivatizing cleaved end nucleotides in each fragment with the other
member of the specific binding pair,
(c) cleaving the derivatized DNA fragments with a second restriction enzyme
to produce short DNA fragments,
(d) binding the short fragments to the immobilized member, leaving a free
end nucleotide on each derivatized fragment,
(e) separating the derivatized fragments from the non-derivatized
fragments,
(f) attaching a reporter to one of the free end nucleotides in each
derivatized shorter fragment,
(g) separating the reporter labeled, separated fragments from the solid
support, and
(h) fractionating the reporter labeled fragments according to size.
25. The method set forth in claim 24 which includes the step of: analyzing
the fractionated fragments for the presence of labeled fragments as a
function of size.
26. The method of claim 25 wherein the reporter is nucleotide specific.
27. The method of claim 24 wherein the reporter is nucleotide specific.
28. The method of claim 27 wherein the solid support is beads that are
water insoluble and stable to the physical and chemical conditions of
steps d, e, f and g of claim 24.
29. The method of claim 28 wherein the beads are chromium dioxide.
30. The method of claim 29 wherein the beads are separated from their
surrounding environment by a magnetic field.
31. The method of claim 26 wherein the solid support is beads that are
water insoluble and stable to the physical and chemical conditions of
steps d, e, f and g of claim 24.
32. The method of claim 31 wherein the beads are chromium dioxide.
33. The method of claim 28 wherein the step of subjecting the short
fragments to the immobilized member to attach the derivatized short
fragments to the beads includes the step of separating the beads with
attached derivatized short fragments from the non-derivatized fragments.
34. The method of claim 33 wherein the beads are chromium dioxide.
35. The method of claim 34 wherein the step of separating the beads is
accomplished by the use of a magnetic field.
36. The method of claim 24 wherein the free end nucleotide is on the 3'
strand of DNA.
37. A method for mapping DNA segments made up of duplex strands of
nucleotides using specific binding substances (one and the other), one
substance being immobilized on a solid support, by the steps of:
cleaving each segment with a first restriction enzyme to produce fragments
of DNA each having a cleaved end,
attaching the other substance to the cleaved ends to provide anchor ends,
cleaving the DNA fragments with a second restriction enzyme to provide
shorter DNA fragments and free ends,
separating shorter fragments with an anchor end from the remaining shorter
fragments by binding the anchor ends to the one substance,
attaching reporter labeled nucleotides to the free end of each separated
shorter fragment,
removing the separated shorter fragments from the one substance,
fractionating the removed shorter fragments.
38. A method for mapping DNA segments made up of duplex strands of
nucleotides using specific binding pairs, one member of the pair being
immobilized on a solid support, by the steps of:
(a) cleaving each segment with a first restriction enzyme to produce
fragments of DNA each having a cleaved end,
(b) derivatizing cleaved end nucleotides in each fragment with the other
member of the specific binding pair,
(c) cleaving the derivatized DNA fragments with a second restriction enzyme
to produce short DNA fragments, the second cleaved ends being
non-derivatized,
(d) attaching a reporter to each of the non-derivatized ends of the short
fragments,
(e) binding the short fragments with derivatized ends to the one member,
(f) separating the bound, derivatized fragments, some of which are
reporter-labeled at one end, from the fragments that are not derivatized
at either end,
(g) separating the bound, reporter-labeled fragments from the solid
support,
(h) fractionating the reporter labeled fragments according to size.
39. A method for mapping DNA segments made up of duplex strands of
nucleotides using specific binding pairs, one member of the pair being
immobilized on a solid support, by the steps of:
(a) cleaving each segment with a first restriction enzyme to produce
fragments of DNA each having a cleaved end,
(b) derivatizing cleaved end nucleotides in each fragment with the other
member of the specific binding pair,
(c) cleaving the derivatized DNA fragments with a second restriction enzyme
to produce short DNA fragments, binding the derivatized fragments to the
one member,
(d) cleaving the bound DNA fragments with a second restriction enzyme to
produce short DNA fragments, some of which have one bound, derivatized and
one free end and some of which are not derivatized at either end,
(e) separating the bound short fragments from the unbound short fragments,
(f) attaching a reporter to one of the free end nucleotides in each bound
short fragment,
(g) separating the reporter-labeled, bound fragments from the solid
support,
(h) fractionating the reporter labeled fragments according to size. |
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Claims |
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Description |
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FIELD OF THE INVENTION
This invention relates to a method for relating DNA segments to each other
by comparing a limited number of polynucleotide fragments which make up
each segment.
BACKGROUND OF THE INVENTION
It often is desirable in molecular biology to determine the relatedness of
DNA segments. Such determinations at the nucleotide sequence level have
many uses in the detection and molecular analysis of DNAs from different
organisms and in the construction of physical and genetic maps. The most
precise method for comparing segments of DNA is to determine the entire
nucleotide sequence of each segment. For large DNA segments, sequencing
becomes prohibitively time-consuming and expensive. Thus, at the present
time, it is not practical to use extensive sequencing to compare DNA
segments when the segments are large or when a large number of segments
are being compared.
Restriction enzymes provide a tool to rapidly analyze DNA segments to
obtain a limited amount of sequence information. Each restriction enzyme
recognizes a specific sequence of DNA, normally four to eight nucleotide
pairs in length, and cleaves DNA at or near this recognition sequence.
Digestion of a DNA segment with a particular restriction enzyme thus
generates a characteristic array of fragments. Typically, these fragments
are separated according to length by electrophoresis through an
appropriate gel matrix. The sizes of the fragments are dependent on the
exact sequence recognized by the restriction enzyme and the spatial
distribution of the recognition sequence within the DNA segment. Thus,
cleavage of a DNA segment with a restriction enzyme indicates that a
particular short recognition sequence is present; the number of fragments
produced indicates how many times the recognition sequence occurs; and the
sizes of the fragments indicate the distance, in nucleotides, between
adjacent recognition sites.
The relatively simple steps involved in digesting DNA with restriction
enzymes and in electrophoresing DNA fragments have made
restriction-fragment analysis a routine method for characterizing and
comparing DNA segments. If two segments of DNA have restriction fragments
of the same length, then there is an increased likelihood that the
segments are similar in sequence or overlapping. The greater the number of
restriction fragments in common, the higher the probability that any two
DNA segments are related. Two procedures have been described that
demonstrate the utility of using restriction-fragment comparisons to
determine the relatedness of a large number (5000-10,000) of DNA segments.
These two procedures are the global mapping method described by Olson et
al. [Proc. Natl. Acad. Sci. U.S.A. 83:7826-7830 (1986)] and the
fingerprint mapping method described by Coulson et al. [Proc. Natl. Acad.
Sci. U.S.A. 83:7821-7825 (1986)].
The first step in the global mapping method is to digest each DNA segment
with a restriction enzyme or combination of restriction enzymes to
generate a collection of restriction fragments. (In the example presented
by Olson et al., each DNA segment was digested with a combination of
HindIII and EcoRI to generate fragments with an average size of 1200 bp.)
Each restriction digest is electrophoresed in a separate lane through an
agarose gel in order to separate fragments according to length. The DNA
restriction fragments are visualized by staining each gel with ethidium
bromide and photographing the gel using ultra violet illumination. The
size of each restriction fragment is determined by comparing its
electrophoretic mobility with the mobilities of known size standards that
were electrophoresed in a parallel lane of each gel. Thus, each DNA
segment is characterized by a list of restriction fragment sizes. A data
base is constructed that contains fragment-size lists for all the DNA
segments being compared. With the aid of a computer program, the
fragment-size lists are compared in a pairwise manner in order to
determine the number of fragments of common size. DNA segments with a
significant number of overlaps are considered to be related. In this
manner related DNA segments spanning regions greater than 100,000 bp can
be identified.
The Olson et al. procedure is referred to as a global mapping method
because almost all the fragments produced in the restriction digest are
used in the construction of the fragment-size lists. The inclusion of
nearly all fragments requires the use of a separation method that can
resolve fairly large fragments, such as electrophoresis through an agarose
gel. Although the use of an agarose gel allows analysis of large
fragments, the ability to discriminate and accurately size closely-spaced
fragments on an agarose gel is somewhat limited. This problem is addressed
in the fingerprint mapping method of Coulson et al. by reducing the size
of the fragments being analyzed to approximately 1000 nucleotides or
smaller. Fragments of this size can be resolved with single base
resolution on a denaturing acrylamide gel.
In the fingerprint mapping method of Coulson et al., each DNA segment is
first cleaved with a restriction enzyme that leaves a 5' overhang. The
ends of these fragments are labeled by incubation with a DNA polymerase in
the presence of a radioactive nucleotide. These radioactively-labeled
fragments are then digested with a second restriction enzyme that cleaves
quite frequently to generate fragments that are now fairly short in length
(average size approximately 200 bp). Each collection of DNA fragments is
then separated according to length by electrophoresis through a denaturing
polyacrylamide gel. Although each sample may contain a large number of
different fragments, only those fragments that have an end generated by
cleavage with the first restriction enzyme are radioactively labeled. The
locations of these labeled fragments on the gel are detected by
autoradiography. The sizes of the detected fragments are determined by
comparison to the mobilities of known size standards. As in the global
mapping method, fragment-size lists are compared in order to determine
which DNA segments are related. Coulson et al. were able to identify
clusters of related DNA segments that spanned regions 35,000 to 350,000 bp
in size.
The global mapping method, the fingerprint mapping method, and other
similar methods use a fragment-size list to characterize the identity of
each DNA segment being examined. Each fragment in the fragment-size list
represents one bit of information that can be used in comparing the
relatedness of DNA segments. One disadvantage of these methods is that the
amount of information about each DNA segment is limited to the number of
fragments in the fragment-size list. If the fragments could be
differentiated in some other way besides just size, more information would
be available for making comparisons. Increasing the information content of
each fragment in the fragment-size list provides better discrimination in
deciding which overlaps between DNA segments are significant.
Another disadvantage of both the global and fingerprint mapping methods is
that a number of steps are required after electrophoresis in order to
obtain digital information that can be used in making comparisons. In the
global mapping method gels must be stained with ethidium bromide and
photographed in order to record the location of each DNA fragment in the
gel. In the fingerprint mapping method gels must be exposed to X-ray film
and the X-ray film must be developed in order to obtain a record of the
mobility of each DNA fragment. In both cases the photographs or
autoradiograms must be analyzed in order to digitize the mobility
information. These manual manipulations increase the time and effort
required to perform the mapping procedures.
SUMMARY OF THE INVENTION
Many of the problems associated with the prior mapping methods are overcome
by the methods of this invention. A method is described for rapidly
characterizing and mapping DNA segments according to a modification of the
Coulson et al. fingerprint mapping method where restriction fragments are
differentially labeled by attachment of nucleotide-specific reporter
molecules. The advantage of differential labeling is that it eliminates
the reliance on fragment size as the sole criterior for rapidly
classifying restriction fragments. Differential labeling is achieved by
first cleaving a DNA segment made up of duplex strands of nucelotides with
a first restriction enzyme or enzymes to produce fragments with an
overhang of nucleotides at the cleaved ends. Although it may be an exact
end restriction enzyme, preferably, the restriction enzyme is one of a
group of ambiguous-end restriction enzymes that generate DNA molecules
with a 5' overhang strand and a 3' recessed strand. Next, a reporter
specific for each nucleotide in each overhang is attached to the 3'
recessed strand of the fragment ends. Following cleavage of the fragments
with a second restriction enzyme to produce short DNA fragments, the short
fragments are separated according to size and analyzed for the presence of
reporters. Thus, each labeled short fragment is characterized not only by
its size, but also by the type of reporter attached.
Alternatively, the primary restriction enzyme or enzymes may be from a
group of restriction enzymes that generate DNA molecules with a 3'
overhang strand (and a 5' recessed strand) or from a group of restriction
enzymes which generate DNA molecules with blunt ends. Although exact end
restriction enzymes may be used, in each case the use of ambiguous-end
restriction enzymes is preferred. In order to label 3' overhangs and blunt
ends, use is made of the 3' exonuclease activity inherent in some DNA
polymerases to remove 3' nucleotides at each cleaved end, thus converting
each 3' overhang or blunt end into a 5' overhang. As part of the same
reaction, the DNA polymerase attaches to each 3' end a reporter
complementary to each nucleotide in the newly created 5' overhang. Thus,
5', 3' and blunt end restriction enzymes, either ambiguous or exact end,
may be used in the gene mapping methods of this invention.
The use of the nucleotide-specific reporters allows the nucleotide sequence
at the labeled end of each restriction fragment to be determined. This
terminal sequence provides another criterion for comparing DNA fragments
other than size. Also, knowledge of each terminal sequence is advantageous
because it provides information about the order of restriction fragments
within the parent DNA segment. Cleavage of DNA with an ambiguous-end
restriction enzyme produces ends with overhangs of complementary sequence.
Thus, if two fragments have a complementary terminal sequence, they are
likely adjacent; and, conversely, if two fragments do not have
complementary terminal sequences, they cannot be adjacent. This
rudimentary order information is useful in comparing the relatedness of
different DNA segments and in mapping the location of restriction sites in
a single DNA segment.
A DNA sequencer using suitable nucleotide reporters, preferably the DNA
sequencer Genesis 2000.TM. sold by E. I. du Pont de Nemours and Company,
Wilmington, Del. 19898, an instrument capable of detecting DNA fragments
having fluorescent reporters, makes it possible readily to add sequence
information to the size information being compared in the fingerprint
mapping method. The use of the labeled dideoxynucleotides in this
invention takes advantage of certain restriction enzymes that cleave DNA
leaving ends or overhangs containing bases which are not included in the
restriction enzyme recognition specificity.
In an alternative method of this invention, the versatility of the above
described methods are greatly enhanced by attaching the nucleotide
specific reporters to the secondary cleaved end after the second cleavage.
This permits virtually any restriction enzyme to be used for the primary
cleavage. Also by attaching the reporter after the second cleavage, larger
DNA molecules (typically those 50 kilobases and larger) may be readily
mapped. The problem occurring with large DNA molecules is that it is
necessary to separate the subset of labeled fragments from the remainder
of the DNA to prevent distortion of the DNA fragment pattern caused by
overloading the size fractionating gel.
According to this alternative method, specific binding substances (e.g.,
one member a ligand and the other member a receptor) or pairs are used.
One member (the ligand) is attached to the cleaved ends of the primary
cleavage fragments. These ends will be referred to hereinafter in this
discussion as the "anchor ends". Next the DNA fragments are cleaved a
second time using a different restriction enzyme to provide shorter
fragments. The shorter fragments with an anchor end are separated from the
remainder of DNA fragments. This may be accomplished by the use of a solid
support coated with or secured on the outside with the other member
(receptor). The shorter fragment anchor ends bind to the other member of
the binding pair on the solid support. So bound, the solid support is
separated from the unbound DNA fragments and reporter labeled
nucleotide(s) are attached to the free end of each of the separated
shorter fragments. The labeled shorter fragments are removed from the
solid support and fractionated. Each fragment size and reporter identity
are recorded for use in the mapping procedure.
The end result of these procedures is to characterize a DNA segment with a
list of fragments where each fragment is identified by both its size and
its 3' terminal bases. Fragment lists of different DNA segments can be
compared, searching for fragments that are identical in size and terminal
base identity. A significant number of matching fragments indicates that
two DNA segments are related. Inclusion of the 3' terminal base(s)
identity adds significantly to the amount of information in each fragment
list. This means that comparisons of DNA segments can be accomplished more
rapidly and accurately than with lists that rely solely on fragment size.
Thus, if fragments from large genomes are being compared, the methods of
this invention considerably enhances the confidence with which overlaps
can be determined.
DESCRIPTION OF THE DRAWINGS
The present invention will be better understood by considering the Examples
in connection with the accompanying drawings in which:
FIG. 1 is a plot of photomultiplier tube output as the ordinant and time as
the abscissa depicting fluorescently labeled DNA fragments from the Becker
strain of pseudorabies virus; and
FIG. 2 is a plot of photomultiplier tube output as the ordinant and time as
the abscissa depicting fluorescently labeled DNA fragments from the Bartha
strain of pseudorabies virus.
DETAILED DESCRIPTION OF THE PREFERRED METHODS
The method of this invention involves mapping DNA in a manner which
provides information not only as to the size of DNA fragments but also
information as to which nucleotides are on the ends of each fragment. It
provides a means to rapidly characterize DNA segments by attaching
distinguishable nucleotide-specific reporters to restriction fragment
ends. These characterizations can be used to compare different DNA
segments for relatedness and to help determine restriction maps, that is,
the spatial distribution of restriction sites along a DNA segment.
To implement the method, restriction enzymes are used to cleave the
phosphodiester backbone in each strand of double-stranded DNA. Depending
on the particular restriction enzyme, this cleavage results in a pair of
complementary cleaved ends in which, for each cleaved end, one strand of
DNA has a 5' overhang and the other DNA strand has a 3' recess (also it
can result in a blunt end, or a 3' overhang). An example of a 5' overhang
is shown by the structure:
##STR1##
The number and identity of the nucleotides in the 5' overhang depends on
the particular restriction enzyme. For the restriction enzymes discovered
so far, the number of nucleotides in the 5' overhang ranges from one to
five.
The 5' overhangs of one DNA strand generated by many restriction enzymes
are suitable substrates for DNA polymerases. In the presence of the four
natural deoxyribonucleoside triphosphates (dNTP's), a DNA polymerase will
repeatedly attach nucleotides to the recessed 3' hydroxyl group of the
other strand until it fills in the length of the 5' overhang, resulting in
a blunt-ended fragment. The nucleotides added to the 3' recessed end are
complementary to the nucleotides in the 5' overhang. If one or all the
dNTP's are replaced with reporter-containing analogues, then a DNA
polymerase will attach these reporters to the free 3' hydroxyl group,
resulting in an end-labeled restriction fragment. If the reporters are
nucleotide-specific, then restriction fragments can be differentially
labeled with reporters complementary to the nucleotides in the 5'
overhang.
The restriction enzymes that generate 5' overhangs can be grouped into two
subclasses. For the first type of enzyme, the nucleotides in the 5'
overhang are exactly defined. These will be referred to as 5'-exact-end
restriction enzymes. For the second type of enzyme, the 5' overhang can
contain several possible nucleotide combinations. These will be referred
to as 5'-indeterminate-end or 5'-ambiguous-end restriction enzymes. In
order to achieve nucleotide-specific differential labeling of restriction
fragments, the preferred approach is to use 5'-ambiguous-end restriction
enzymes. Below is a list of known 5'-ambiguous-end restriction enzymes
that could be used in this application and the ends generated by cleavage
with each enzyme. N denotes that any base (A,C,G, or T) can be present. In
some cases, more than one enzyme will produce the same type of end. These
are known as isoschizomers. Only one member of each isoschizomer family is
listed.
______________________________________
AccI
##STR2##
##STR3## M = A or C K = G or T
AflIII
##STR4##
##STR5## R = A or G
AvaI
##STR6##
##STR7## Y = C or T R = A or G
AvaII
##STR8##
##STR9## W = A or T
BanI
##STR10##
##STR11## Y = C or T R = A or G
BbvI
##STR12##
##STR13##
BinI
##STR14##
##STR15##
BspMI
##STR16##
##STR17##
BstEII
##STR18##
##STR19##
BstNI
##STR20##
##STR21## W = A or T
DdeI
##STR22##
##STR23##
Eco0109
##STR24##
##STR25## R = A or G Y = C or T
EcoRII
##STR26##
##STR27## W = A or T
Fnu4HI
##STR28##
##STR29##
FokI
##STR30##
##STR31##
HgaI
##STR32##
##STR33##
HinfI
##STR34##
##STR35##
MaeIII
##STR36##
##STR37##
MstII
##STR38##
##STR39##
NciI
##STR40##
##STR41## S = G or C
PpuMI
##STR42##
##STR43## R = A or G, Y = C or T W = A or T
RsrII
##STR44##
##STR45## W = A or T
Sau96I
##STR46##
##STR47##
ScrFI
##STR48##
##STR49##
SfaNI
##STR50##
##STR51##
StyI
##STR52##
##STR53## W = A or T
TthlllI
##STR54##
##STR55##
______________________________________
The labeled restriction fragments are digested a second time with a
different restriction enzyme to generate still shorter fragments. The
shorter fragments are then size separated, typically by gel
electrophoresis, and the reporters on the separated fragments detected.
This provides both size and nucleotide sequence information for each
separated fragment which greatly facilitate mapping and comparison of
fragments for similarities as described.
By way of illustration, the execution of this method, using preferred
fluorescent reporters for the application of an enzyme leaving restriction
fragments with a 5' overhang, requires four steps.
Step 1-DNA segments are cleaved to generate restriction fragments with 5'
overhangs by incubating each DNA sample under the appropriate conditions
with a restriction enzyme or combination of restriction enzymes,
preferably one or more of the 5'-ambiguous-end restriction enzymes. (At
this point, the restriction fragments may be purified by ethanol
precipitation or some other means, but this is generally not necessary.)
Step 2-Nucleotides with nucleotide specific reporters, complementary to the
nucleotide or nucleotides in each 5' overhang, are attached to the
recessed 3' ends of the restriction fragments. While any reporters can be
used, such as those sold by Applied Biosystems, Inc., Foster City, Calif.,
fluorescent reporters of the type sold for use in the Du Pont Genesis.TM.
DNA sequencer are preferred. These reporters have similar stabilities and
do not affect the electrophoretic separation characteristic of the
nucleotides they are attached to. The preferred reporters are one of a set
of chain terminators, more specifically the flourescence-labeled
2',3'-dideoxynucleoside triphosphates (F-ddNTP's):
7-(SF505-Sar-AP3)ddc7GTP [15],
7-(SF512-Sar-AP3)ddc7ATP [14],
5-(SF519-Sar-AP3)ddCTP [13], and
5-(SF526-Sar-AP3)ddTTP [12].
(The compound numbers refer to the structures shown below)
The preferred method for attaching reporters is to incubate the collection
of restriction fragments under the appropriate conditions with a DNA
polymerase and a mixture of F-ddNTP's and unlabeled dNTP's. At each 3'
restriction fragment end, this incubation results in the attachment of a
distinctive fluorescence-labeled dideoxynucleotide complementary to each
nucleotide in the 5' overhang. The selection of DNA polymerase depends
upon the reporter substrate being attached. For the F-ddNTP's, reverse
transcriptase and phage T7 DNA polymerase are appropriate DNA polymerases.
The relative concentrations of F-ddNTP's and unlabeled dNTP's depends upon
the number of reporters to be attached to each restriction fragment end.
If it is desired to attach only one reporter per end, then only F-ddNTP's
are included in the incubation. If the 5' overhangs consist of more than
one nucleotide and it is desired to attach more than one reporter at the
3' recessed strand end of each, then unlabeled dNTP's are also included.
At each possible addition point on the recessed strand, either a F-ddNTP
is added and the chain is terminated and labeled, or a dNTP is added and
the chain is now a substrate for further addition. The F-ddNTP and dNTP
concentrations are adjusted to give a suitable distribution of labeled
fragments, differing in length by one nucleotide. After the labeling
incubation, the DNA polymerase is inactivated by incubation at elevated
temperature or some other means. (The labeled fragments can be purified by
ethanol precipitation or some other means).
Step 3-The labeled fragments are next digested with a second restriction
enzyme or combination of restriction enzymes in order to generate shorter
fragments. Any restriction enzymes can be used at this point as long as
they are different than the restriction enzymes used in the primary
cleavage. This secondary cleavage serves two purposes. First, after the
labeling reaction, both ends of each DNA fragment are labeled and this
double-labeling would interfere with effective discrimination of the
nucleotide-specific reporters. Thus, it is desirable to use a secondary
cleavage to generate shorter fragments so that, in general, each labeled
end is on a separate fragment of distinct size. Second, the generation of
shorter fragments allows the use of separation procedures that can achieve
single-base resolution, such as electrophoresis through denaturing
polyacrylamide gels. (Again, the DNA fragments can be purified by ethanol
precipitation or some other means.)
Step 4-The DNA fragments present after the secondary cleavage reaction are
separated according to size and analyzed for the presence and identity of
nucleotide-specific reporters. The preferred method is to use the Du Pont
Genesis 2000.TM. gel electrophoresis and detection system. Other
fluorescence detector and nucleotide-specific reporters may be used as
desired. The reporters need not be fluorescent. With the Du Pont system,
the time it takes for a labeled fragment to reach the detection zone is a
measure of that fragment's mobility through the gel. By comparing this
mobility data to the mobility data of known size standards, the size of
each labeled fragment can be determined. Within the detection zone, the
DNA fragments are irradiated by a laser beam and excitation/emission of
the fluorescent reporters occurs as the fragments move through the zone.
Using appropriate filters and a dual-detector system, each of the four
nucleotide-specific reporters can be identified on the basis of their
distinctive emission spectra.
In another alternative embodiment step 2 of the above-identified method may
be modified so that the labeling can occur also with DNA fragments
generated by the 3' overhang or blunt end restriction enzymes. In each
case the restriction enzymes may be exact end or preferably ambiguous end.
To facilitate the use of these additional enzymes, the invention exploits
the 3'-exonuclease activity inherent in some DNA polymerases, such as, the
Klenow fragment of DNA polymerase I or T7 DNA polymerase or T4 DNA
polymerase. When presented with a blunt end or a 3' overhang, these
enzymes will remove 3' nucleotides until a 5' overhang is generated. In
the absence of deoxynucleoside triphosphates, the enzyme will continue to
remove 3' nucleotides creating longer and longer 5' overhangs. In the
presence of deoxynucleoside triphosphates, the enzymes adds nucleotides
back to the 3' end to generate blunt-ended fragments. The nucleotides
added are complementary to the bases in the opposite strand. Thus, blunt
ends and 3' overhang ends can be labeled with nucleotide-specific
reporters by using the appropriate DNA polymerase and tagged or reporter
labeled nucleotides that are accepted by the enzyme. This means that, in
the mapping procedure presented here, restriction enzymes that generate
blunt ends or 3' overhang ends can be used to cleave the DNA to produce
the ends that will be labeled in a nucleotide-specific manner. The mapping
procedure is more informative if there is some sequence ambiguity at the
ends that are labeled. Thus, in addition to the 5'-ambiguous-end
restriction enzymes listed previously, the following ambiguous-blunt-end
and 3'-ambiguous-end restriction enzymes are especially useful for this
mapping procedure:
__________________________________________________________________________
BglI
##STR56##
##STR57##
BstXI
##STR58##
##STR59##
Eco571
##STR60##
##STR61##
GsuI
##STR62##
##STR63##
HaeII
##STR64##
##STR65## R = A or G Y = C or T
HincII
##STR66##
##STR67## R = A or G Y = C or T
HphI
##STR68##
##STR69##
MboII
##STR70##
##STR71##
MnlI
##STR72##
##STR73##
NlaIII
##STR74##
##STR75##
NlaIV
##STR76##
##STR77##
NspHI
##STR78##
##STR79## R = A or G Y = C or T
PflMI
##STR80##
##STR81##
SfiI
##STR82##
##STR83##
TthlllII
##STR84##
##STR85##
R = A or G Y = C or T
XmnI
##STR86##
##STR87##
__________________________________________________________________________
ENHANCED MAPPING PROCEDURE USING SPECIFIC BINDING SUBSTANCES
In an alternative embodiment of this invention, nucleotide specific
reporters are attached to the cleaved ends produced by the secondary
cleavage. This is true whether 5', 3' or blunt end, ambiguous or
exact-end, restriction enzymes are used. This has the advantage that
virtually any restriction enzyme may be used for the primary cleavage. It
also facilitates the use of this method with larger DNA molecules, i.e.,
specifically those greater than 50 kilobases. The problem occurring with
large DNA molecules is that it is necessary to separate the subset of
labeled fragments from the remainder of the DNA to prevent distortion of
the DNA fragment pattern caused by overloading the size fractionating gel.
According to this method one member of a specific binding pair is attached
to the primary cleaved DNA fragments. For simplicity's sake the cleaved
ends to which the one member is attached will be referred to as the
"anchor ends". Next, the fragments are cleaved a second time to provide
shorter fragments using a different restriction enzyme. The shorter
fragments with the anchor ends are separated from the remainder of the DNA
fragments by the use of a solid support having the other member (receptor)
of the specific binding pair attached to or bound to a solid support,
preferably a bead, as will be described hereinafter. A reporter is
attached to the free (unattached) end of each shorter separated fragment,
separated from its solid support, and finally fractionated according to
size with the size and reporter identity being recorded for each shorter
fragment.
SOLID SUPPORTS
Many different types of solid supports, although beads are preferable, can
be used in this invention. If beads are used, they must be water insoluble
and stable to the physical and chemical conditions to which they are
subjected during linking of one member (receptor) of the specific binding
substance (e.g., avidin) and during elution of the labeled DNA. They must
also be capable of being covalently linked to the specific binding
substance in a manner which is stable to the elution conditions. It is
desirable that the beads exhibit low nonspecific adsorption of nucleic
acids under the binding conditions. The beads must be capable of being
separated readily from the aqueous medium following binding of DNA
fragments, e.g., by settling, centrifugation or application of magnetic
field. Beads in the size range 1-300 microns are satisfactory for
separation by settling or centrifugation. Beads in the size range 10-100
microns are preferred. In general, beads which have been used heretofore
in affinity purifications by hybridization of desired nucleic acids, as
described in Moss et al., J. Biol. Chem., 256:12655-8 (1981), Langda | | |
