U.S. patent number 5,202,231 [Application Number 07/723,712] was granted by the patent office on 1993-04-13 for method of sequencing of genomes by hybridization of oligonucleotide probes.
Invention is credited to Radomir B. Crkvenjakov, Radoje T. Drmanac.
United States Patent |
5,202,231 |
Drmanac , et al. |
April 13, 1993 |
**Please see images for:
( Certificate of Correction ) ** |
Method of sequencing of genomes by hybridization of oligonucleotide
probes
Abstract
The conditions under which oligonucleotides hybridize only with
entirely homologous sequences are recognized. The sequence of a
given DNA fragment is read by the hybridization and assembly of
positively hybridizing probes through overlapping portions. By
simultaneous hybridization of DNA molecules applied as dots and
bound onto a filter, representing single-stranded phage vector with
the cloned insert, with about 50,000 to 100,000 groups of probes,
the main type of which is (A,T,C,G)(A,T,C,G)N8(A,T,C,G),
information for computer determination of a sequence of DNA having
the complexity of a mammalian genome are obtained in one step. To
obtain a maximally completed sequence, three libraries cloned into
the phage vector, M13, bacteriophage are used: with the 0.5 kb and
7 kbp insert consisting of two sequences, with the average distance
in genomic DNA of 100 kbp. For a million bp of genomic DNA, 25,000
subclones of the 0.5 kbp are required as well as 700 subclones 7 kb
long and 170 jumping subclones. Subclones of 0.5 kb are applied on
a filter in groups of 20 each, so that the total number of samples
is 2,120 per million bp. The process can be easily and entirely
robotized for factory reading of complex genomic fragments or DNA
molecules.
Inventors: |
Drmanac; Radoje T. (Beograd,
YU), Crkvenjakov; Radomir B. (Beograd,
YU) |
Family
ID: |
27390498 |
Appl.
No.: |
07/723,712 |
Filed: |
June 18, 1991 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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175088 |
Mar 30, 1988 |
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Foreign Application Priority Data
Current U.S.
Class: |
435/6.18;
435/6.1; 436/501; 436/94; 536/23.1; 536/24.32 |
Current CPC
Class: |
C12Q
1/6827 (20130101); C12Q 1/6874 (20130101); C12Q
1/6874 (20130101); C12Q 1/6827 (20130101); C12Q
1/6874 (20130101); C12Q 2565/518 (20130101); C12Q
2565/518 (20130101); C12Q 2565/518 (20130101); C12Q
2525/204 (20130101); Y10T 436/143333 (20150115) |
Current International
Class: |
C12Q
1/68 (20060101); C12Q 001/68 (); C07H 015/12 ();
G01N 033/566 (); G01N 033/48 () |
Field of
Search: |
;435/6 ;536/26,27,28,29
;436/501,94 ;935/77,78 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Chan et al., Nuc. Acids Res. 13(22):8083-8091 (1985). .
Maniatis et al., Molecular Cloning A Laboratory Manual Cold Spring
Harbor Lab., N.Y. 1982. .
Poustka et al., Cold Spring Harb. Symp. Quant. Biol. LI:131-139
(1986)..
|
Primary Examiner: Moskowitz; Margaret
Assistant Examiner: Zitomer; Stephanie W.
Attorney, Agent or Firm: Marshall, O'Toole, Gerstein, Murray
& Bicknell
Parent Case Text
This application is a continuation of application Ser. No.
07/175,088 filed Mar. 30, 1988, abandoned.
Claims
We claim:
1. A method of sequencing a nucleic acid fragment, comprising:
(a) contacting a nucleic acid fragment with a set of
oligonucleotide probes of predetermined sequence and length under
hybridization conditions that allow differentiation between (i)
those probes of said set which are exactly complementary to part of
said fragment and (ii) those probes of said set which are not
exactly complementary to part of said fragment;
(b) detecting those probes of said set which are exactly
complementary to part of said fragment; and
(c) determining the sequence of said fragment from the subset of
those probes of said set which are detected as exactly
complementary to part of said fragment by compiling their
overlapping sequences.
2. The method of claim 1, wherein said oligonucleotide probes are
8-mers to 20-mers.
3. The method of claim 1, wherein said oligonucleotide probes are
11-mers.
4. The method of claim 2, wherein each oligonucleotide probe is an
11-mer containing within it a unique 8-mer, such that each 11-mer
is characterized by the designation (N2)N8(N1) or (N1)N8(N2)
wherein N2 represents either two nucleotides at the 5'-terminal or
the 3'-terminal of said probe; N1 represents either the 3'-terminal
nucleotide or the 5' terminal nucleotide of said probe; and N8
represents said unique 8-mer.
Description
TECHNICAL FIELD
The subject of this invention belongs to the field of molecular
biology.
TECHNICAL PROBLEM
Genomes range in size from about 4.times.10.sup.6 base pairs (bp)
in E. coli to 3.times.10.sup.9 bp in mammals. Determination of the
primary structure, i.e., sequence, of the entire human genome, is a
challenge of the 20th century. A further challenge for biology is
the determination of the entire genomic sequence for characteristic
species of the living world. It would allow qualitative progress in
explaining the function and evolution of organisms. It would also
be a great step forward in the explanation and treatment of many
diseases, in the food industry and in the entire field of
biotechnology.
STATE OF THE ART
Prior Art
Recombinant DNA technology has allowed the multiplication and
isolation of short fragments of genomic DNA (from 200 to 500 bp)
whereby a sufficient quantity of material for determination of the
nucleotide sequence may be obtained in a cloned fragment. The
sequence is determined on polyacrylamide gels which separate DNA
fragments in the range of 1 to 500 bp, differing in length by one
nucleotide. Distinguishing among the four nucleotides is achieved
in two ways: (1) by specific chemical degradation of the DNA
fragment at specific nucleotides, in accordance with the Maxam and
Gilbert method (Maxam, A. M. and Gilbert, W., 1977, Proc. Natl.
Acad. Sci., 74, 560); or (2) utilizing the dideoxy sequencing
method described by Sanger (Sanger, F., et al., 1977, Proc. Natl.
Acad. Sci., 74, 5463). Both methods are laborious, with competent
laboratories able to sequence approximately 100 bp per man per day.
With the use of electronics (computers and robots), sequencing can
be accelerated by several orders of magnitude. The idea of
sequencing the whole human genome has been discussed at many
scientific meetings in the U.S.A. (Research News, 1986, Science,
232, 1598-1599). The general conclusion was that sequencing is
possible only in big, organized centers (a sequencing factory) and
that it would take about 3 billion dollars and at least ten years.
For the time being the Japanese are the most advanced in organizing
components of one such center. Their sequencing center has the
capacity of about 1 million bp a day at the price of about 17.cent.
per bp (Commentary, 1987, Nature, 325,771-772). Since it is
necessary to sequence three lengths of a genome, because of random
formation of cloned fragments of about 500 bp, 10 billion bp could
be sequenced in approximately 30 years in such a center, i.e., it
would take 10 such centers to sequence the human genome in several
years.
DESCRIPTION OF THE INVENTION
Our process of sequencing, compared with the existing ones,
involves an entirely different logic, and is applicable
specifically for determining a sequence of complex DNA fragments
and/or molecules (more than 1 million base pairs). It is based upon
specific hybridization of oligonucleotide probes (ONPs), having a
length of 11 to 20 nucleotides.
Conditions of hybridization of ONPs have been found under which
complete homology with the target sequence is differentiated from a
single base pair mismatch (Wallace, R. B., et al., 1979, Nucleic
Acid Res., 6, 3543-3557). If the method of hybridization with 3M
tetramethyl ammonium chloride is used, the melting point of the
hybrid is dependent only on the ONP length, not its GC content
(Wood, W. I., et al., 1985, Proc. Natl. Acad. Sci. U.S.A., 82,
1585-1588). Thus, hybridization under such conditions unequivocally
determines the sequence. Hybridization of genomic DNA multiplied as
subclones of convenient length with a sufficient number of ONP's
will allow the entire genome to be sequenced with the aid of
computerized assembly of these detected sequences. We believe this
method is an order of magnitude quicker and cheaper than technique
presently being developed. Therefore, it is more suitable for
genomic sequencing of all characteristic species.
In order to apply this method, it is necessary to optimize the
length, sequence and number of ONPs, the length and number of
subclones and the length of the pooled DNA which may represent a
hybridizing sample. Eleven-mer ONPs are the shortest
oligonucleotides that can be successfully hybridized. This means a
priori that 4.sup.11 (4,194,304) ONPs are needed to detect each
sequence. The same number of independent hybridizations would be
necessary for each subclone or a pool of subclones. Positively
hybridizing ONPs would be ordered through overlapping 10-mers. This
results in the DNA sequence of the given subclone.
The process of assembling a subclone sequence is interrupted when
the overlapping 10-mer is repeated in the given subclone. Thus,
uninterrupted sequences are found only between repeated 10-mers or
between longer oligonucleotide sequences (ONSs). These fragments of
a subclone sequence (SF) cannot always be ordered in an unambiguous
linear order without additional information. Therefore, it is
important to determine the probable number of SFs (Nsf) distributed
along certain length of DNA; this can be achieved through the
application of probability calculations.
The distribution of ONSs along a randomly formed DNA sequence is
binomial. The average distance between identical neighboring ONSs
(A) depends only on the ONS length (L), and is given as: A=4.sup.L.
The probability of having ONSs repeated N times in a fragment of
the length of Lf bp is given as:
where C(N,Lf) represents the number of the N class combinations
consisting of Lf elements. The expected number of different ONSs
having the length L or average distance A, which are repeated N
times in a Lf bp fragment, is given as the product P(N,Lf).times.A.
If a sequence is assembled through the overlapping length L or
through average distance Ao, then Nsf in a fragment Lf is
represented as:
If all 11-mers (4.times.10.sup.6) are used, about 3 SFs are to be
expected per Lf of the length of 1.5 kb. We shall return to the
problem of ordering SFs later.
The required synthesis of 4.times.10.sup.6 11-mers, is
impracticable for sequencing by hybridization (SBH). However, it is
unsuitable to omit a significant number of ONPs (more than 25%),
because it leads to gaps in the sequence. A far better way to
decrease the number of independent ONP syntheses and of independent
hybridizations is to use ordered ONP groups. This method requires
sequencing of shorter fragments, but no gaps appear in the
resultant sequence. A forty-fold decrease in the number of
syntheses and hybridizations requires a seven-fold increase in the
number of subclones.
The use of ordered ONP groups, in an informative respect, is the
same as using shorter ONPs. For instance, there are 65,536
different 8-mer ONSs. Since 8-mer ONSs, according to the current
knowledge, cannot form a stable hybrid, the 11-mer group can be
used as an equivalent. In other words, all 11-mers in a group have
one 8-mer in common, so that the information obtained concerns only
its presence or absence in the target DNA. Each of the anticipated
groups of 11-mers contains 64 ONPs of the (N2)N8(N1) type
(5'.fwdarw.3' orientation is as written, /Nx/means x unspecified
bases), and /Ny/ means y specified bases. A sequence can be
detected with about 65,000 such groups. If equation (2) is applied,
then DNA fragments 200 bp long are expected to have 3 SFs on
average. Due to dispersion, some fragments of this length will have
10 and more SFs.
Because of its unrandom GC and dinucleotide composition, ONPs of
the (N2)N8(N1) type are not very convenient for sequencing
mammalian DNA. The more AT bases contained in the common sequence
of an ONP group, the longer it should be. Taking this into account,
there remain three types of suitable probes: (N1)N10, where N10
stands for all 10-mers not containing G+C; (N1)N9(N1), where N9
stands for all 9-mers containing 1 or 2 G+C; and (N2)N8(N1), where
N8 represents all 8-mers containing 3 or more G+C. About 81,000
such ONP groups are necessary. The average value of their Ao(Aao)
is about 30,000. Equal Ao value in random DNA requires about
130,000 ONPs of the (N2)N8(A or T) and (N2)N8(or rG) types. These
ONP groups allow sequencing of fragments 300 bp long, with 3 SFs at
average. This increase of 25% in the number of syntheses allows a
multifold decrease in the number of necessary SCs (see below).
Apart from these probes it is necessary to synthesize an additional
20,000 ONPs in order to (1) solve the problem of repetitive
sequences, (2) confirming the ends of inserts and (3) supplementing
information lost due to the fact that it is unfeasible to use ONP's
which hybridize with vector DNA.
Repetitive sequences, or, generally speaking, ONSs repeated in
tandems and having the length of one or more bp (AAAAAAA . . .
TCTCTCTC . . . TGATGATG . . . ) represent a problem in sequencing
by hybridization. The above mentioned probes cannot determine
length of repetitive sequences that are longer than the common part
of a ONP group. Therefore, the precise determination of repetitive
ONSs up to 18 bp long, that represent the largest part of these
ONPs, requires application of the following ONPs: 160 NP An and Tn,
where the value of n stretches between 11 and 18 bp, 4 ONP (AT),
ONP Cn and G.sub.n where n is valued from 9 to 18 bp, 4 ONP (AT)n
where n takes on the values of (12, 14, 16, 18), 25 ONPs (AC)n,
(AG)n (TC)n (TG)n and (CG)n, where n is valued (10, 12, 14, 16,
18), 60 ONPs of the (N1 N2 N3)n type which encompass all
trinucleotides and n is (12, 15, 18 ), 408 ONPs which include all
5-mers in tandems, having the length of 15 and 18 bp, 672 ONPs
consisting of 6-mer tandems up to 18 bp long, and 2340 ONPs
consisting of 7-mer tandems 18 bp long. The total number of these
ONPs is 3725.
In order to confirm the ends of DNA inserts in a subclone, it is
necessary to synthesize an additional 2048 ONPs of the N6(N5) and
(N5)N6 types, where N6 represents sequences of the vector ends, and
(N5) represents all possible 5-mers in both cases.
The problem of the vector DNA can be solved in two ways. One is
prehybridization with cold vector DNA shortened for 7 bp at both
sides of the cloning site. The other method is to omit ONPs
complementary to the vector DNA. Since phage M13 has been chosen as
the most suitable phage vector (see below), it would eliminate the
use of approximately 7,000 ONPs. This is a significant percentage
(11%) if 65,000 ONP's [(N2)N8(N1)] are utilized. It can be
decreased to about 3% if, instead of the given 7,000 ONPs, an
additional 21,000 ONPs of the (N1)(N.sup.0 1)N8(N1) type are used,
where N8 is 7,000 M13 8-mers, and (N.sup.0 1) represents each of
the trinucleotides not present by the given 8-mer.
The calculations, supra, are related to the sequencing of single
stranded DNA. To sequence double stranded DNA it is not necessary
to synthesize both complementary ONPs. Therefore, the number of
necessary ONPs can be halved. Yet, due to advantages of the M13
system, we will discuss further the method of sequencing the single
stranded DNA. In this case, gaps of unread sequences will appear in
the subclones, because half the ONPs are used. However, a gap in
one subclone will be read in the subclone containing the
complementary chain. In a representative subclone library each
sequence is repeated about 10 times on average. This means that it
is probable to have each sequence cloned in both directions, i.e.,
that it will be read on both DNA strands. This allows the use of
uncomplementary ONPs with only an increase in algorithmic
computation. Thus, the total number of necessary ONPs would be
approximately 50,000. If an M13 vector, able to package both
strands either simultaneously or successively could be devised, the
use of uncomplementary ONPs would not impose any additional
requirement.
All subclone and/or all pools of subclones hybridize with all
anticipated ONPs. In this way a set of positively hybridizing ONPs
is attained for each subclone, i.e., subclone pool. These ONPs are
ordered in sequences by overlapping their common sequences, which
are shorter than the ONPs by only one nucleotide. In order to
detect overlapping ONPs faster, it is necessary to determine in
advance which ONP overlap maximally with each synthesized ONP.
Thus, each ONP will obtain its subset of ONPs: (ONPa, ONPb, ONPc,
ONPd) 5' ONP.times.3' (ONPe, ONPf, ONPg, ONPh). Ordering must
follow the route of detecting which one out of the four ONPs from
the 5' side, and which one out of four from the 3' side positively
hybridize with the given subclone, i.e., pool. Assembling continues
until two positively overlapping ONPs for the last assembled ONP
are found. Thereby, one SF is determined. When all SFs are extended
to the maximum, the process is terminated.
The number of SFs is increased for a larger length of DNA by using
described ONP groups. Generally, unequivocal ordering can be
achieved with 3 SFs per subclone when the SFs are counted in the
same way as Nsf is calculated in equation (2). These SFs are
recognized as the two placed at the ends of an insert with third
placed, logically, in between. The ordering of SFs cannot be solved
for a convenient length of a subclone because it would be too
short. Our solution is the mutual ordering of SFs and large numbers
of subclones, with the possibility of using subclone pools as one
sample of hybridization and/or competitive hybridization of labeled
and unlabeled ONPs.
To obtain the maximum extended sequence by SBH on subclones that
may be used repeatedly, it is necessary to use three subclone
libraries in the M13 vector, with 0.5-7 kb inserts and with inserts
of different size, which consist of two sequences: their distance
in genomic DNA should be about 100 kb. The first library serves
primarily to order SFs. These subclones cannot be preserved for
later experimentation. These subclones enter hybridization as pools
obtained by simultaneous infection during or after growth of a
phage. The second library is the basic one. The subclones it
contains are convenient for further use. The length of 7 kbp
represents the current limit of the size of an insert suitable for
successful cloning in M13. The function of the third library is to
regularly associate parts of sequences separated by highly
homologous sequences longer than 7 kbp as well as uncloned DNA
fragments into an undivided sequence.
Hybridization of subclones of all libraries with ONPs, and
computation of SFs is followed by mutual ordering of the SFs and
subclones. The basic library is the first to be ordered.
Overlapping subclones are detected by the content of the whole or
of a part of starting SFs of the starting subclone. Generally, all
mammalian SFs having a length of 20 bp or more are suitable. The
average SF length of these subclones is calculated on the basis of
equation (2) is 12 bp. This means that there are enough SFs of
suitable length. Besides, it is known among which SFs--most often
there are two--is the one which continues from the starting SF. In
this case both sequences are examined; the correct one is among
them, and overlapping subclones are detected by it. On the basis of
content of other SFs, exact displacement of overlapping subclones
in relation to the starting subclone is determined. The linear
arrangement of subsets (SSFs) is achieved by detecting of all
subclones overlapping with the starting subclone. SSFs are defined
by neighboring ends of overlapping subclones (either
beginning-beginning, beginning-end or end-end). The process of
overlapping subclones is continued with the SF of the jutting SSF
of the most jutting subclone. The process of assembling is
interrupted when encountered with an uncloned portion of DNA, or,
similar to forming of SFs, with a repeated sequence longer than 7
kbp. The maximum size of groups of ordered overlapping subclones 7
bp long is obtained by this method, as well as linear arrangement
of the SSFs of their SFs.
The length of DNA contained in a SSF an fundamental in this
procedure. This length depends on the number of SSFs, which is
equivalent to the number of subclone ends, i.e., two times the
number of subclones. A representative library of DNA fragments of
1,000,000 bp requires 700 SCs of 7 kbp. Therefore, the average size
of a SSF is 715 bp. The real average number of SFs within an SSF is
not 1/10 the number of SFs in a subclone 7 kbp long nor is it
dependent on the length of subclone. Instead, the real number is
dependent on the length of an SSF. According to equation (2), for
the length of 715 bp and an Aao of 30,000 bp (the values for
described ONPs), 16 SFs with an average length of 45 bp are
expected on average. The ordering of SFs within obtained SSFs is
performed through the 0.5 kb subclone library. This method does not
require all subclones to be individual; the use of a subclone pool
is sufficient. The subclones in a respective pool are informative
if they do not overlap with each other. Informatively and
technologically suitable is a 10 kb pool of cloned DNA, although it
is not the limit. The number of such necessary subclones or pools
is such that the maximum size of an SSF formed by them can be no
longer than 300 bp. The ONPs proposed for this length are expected
to give 3 SFs (equation 2), which, as explained, may be
unequivocally ordered. Utilizing binomial distribution has enabled
the derivation of the equation:
where Nsa is the number of SSFs greater than Lms, Nsc is the number
of bp per fragment or molecule of the DNA being sequenced, Lms
stands for the size of an SSF, which an average gives the number of
3 SF, which can be ordered; in this case it amounts 300 bp. On the
basis of this equation it is determined that 25,000 subclones of
0.5 kb are necessary for a DNA fragment 1 million bp long. The
number of 10 kb pools is 1250. Average size of an SSF obtained with
these subclones is 20 bp.
The ordering of SFs is performed by computer detection of pools
containing subclones which overlap with the starting SSF. This
detection is performed on the basis of the content of a part of the
whole SF, randomly chosen from the starting SSF. Contents of other
SFs from the starting SSF determine the overlapping proportion of
subclones of 5 kb. Due to their high density, the arrangement of
SFs in the starting SSF is also determined. At the end of this
process, the sequence of each group of ordered subclones of 7 kb is
obtained, as well as information about which pool contains the
subclone of 0.5 kb bearing the determined sequence. At a certain
small number of loci the sequence will either be incomplete or
ambiguous. Our calculations show that on average it is not less
than one locus per million bp, including 30% of randomly
distributed undetected ONSs. These loci are sequenced by convenient
treatments of subclones which bear them, followed by repeated SBH
or competitive hybridization with suitably chosen pairs of labeled
and unlabeled ONPs or by classical method or by the advanced
classical method.
The procedure of competitive hybridization will be explained by the
example of the sequence 7 bp long repeated twice. In this case two
SFs end and two others begin with the repeating sequence TTAAAGG
which is underlined. ##STR1## Prehybridization with surplus of an
unmarked ONP, e.g., 5'(N2)CATTAAAG(N1)3', which cannot hybridize
with 5'NNCGTTAAAGG 3' due to one uncomplimentary base prevents one
of the labelled ONPs--5'(N2)AAAGGTAC(N1)3' or
5'(N2)AAAGGCCCG(N1)3--from the subsequent hybridization. A pair of
mutually competing probes defines a pair of SFs which follow one
another. This can be confirmed by an alternative choice of a
suitable ONP pair. This procedure may be applied on all repeating
ONSs having the length of up to 18 bp. In order to use it for the
ordering of a multitude of SFs, prehybridization must be separated
from hybridization in both time and space. Therefore, the stability
of a hybrid with unlabelled ONP is important. If such stability
cannot be achieved by appropriate concentrations of ONPs and by
choice of hybridizing temperatures, then a covalent link should be
formed between a cold ONP and complementary DNA by UV radiation in
presence of psoralen. Alternatively, one might use ONPs which carry
reactive groups for covalent linking.
The subclones of the third library are used to link the sequenced
portions into a uniform sequence of the entire DNA fragment being
sequenced. Approximately 170 subclones are required for 1 million
bp. These and other numbers calculated for 1 million bp increase
linearly with length for longer DNA fragments. Since these
subclones contain sequences which are distanced at 100 kbp on
average, they allow jumping over repeated or uncloned sequences,
the size of which increases up to 100 kb. This is done by detecting
which of the two sequenced portions contains sequences located in
one subclone from the library.
The experimental requirement of this method is to have the total
number of 50,000 ONPs and hybridizations, and 2120 separated
hybridizing subclone samples per DNA fragment approximately
1,000,000 bp long.
The libraries described, supra, are made in the phage vector, M13.
This vector allows easy cloning of DNA inserts from 100 to 7,000 bp
long, and gives high titer of excreted recombinant phage without
bacterial cell lysis. If a bacterial culture is centrifuged, a pure
phage preparation is recovered. Additionally, the bacterial
sediment can be used for repeated production of the phage as long
as the bacterial cell pellet is resuspended in an appropriate
nutrient media. With the addition of alkali, DNA separates from the
protein envelope and is simultaneously denatured for efficient "dot
blot" formation and covalent linking to nylon filters on which
hybridization is performed. The quantity of DNA obtained from
several milliliters of a bacterial culture is sufficient to
hybridize one subclone with all the ONPs. A suitable format for
cultivation and robot application on filters are plates similar to
micro-titer plates, of convenient dimensions and number of wells.
Application of DNA subclones on filters is performed by a robot
arm. Even the largest genomes can be satisfactorily sequenced by a
robot arm supplied with 10,000 uptake micropipets. After the DNA
solution has been removed from the plates, the micropipets are
positioned closer to each other by a mechanism reducing the
distance between them to 1 mm. The quantity of DNA suitable for
10,000 subclones is then applied to the filter simultaneously. This
procedure is repeated with the same 10,000 subclones as many times
as necessary. The same procedure is then repeated with all other
subclones, their groups numbering 10,000 each. The number of
"imprints" of one such group with 50,000 ONPs is approximately
1,000, since each filter can be washed and reused 50 times.
The hybridization is performed in cycles. One cycle requires at
maximum one day. All the subclones are hybridized with a certain
number of ONPs in one cycle. In order to have the hybridization
completed within a reasonable period, an experiment in each cycle
should require approximately 1,000 containers, each with one ONP.
For the purpose of saving ONPs, a smaller volume of hybridizing
liquid is used, and therefore filters are added in several turns.
Filters from all hybridizing containers are collected in one
container, and are simultaneously processed further i.e., they are
washed and biotin is used for labelling of ONPs instead of
radioactive particles, colored reactions are developed. All
subclones required for sequencing (up to 10 kb in length) can be
hybridized in containers with the dimensions 20.times.20.times.20
cm without having to repeat individual cycles.
Hybridization sequencing of fragments cloned in plasmid vectors can
be performed one of two ways: by (1) colony hybridization or (2)
"dot blot" hybridization of isolated plasmid DNA. In both cases,
2,000 or 3,000 different ONPs in the vector sequence will not be
utilized, i.e., will not be synthesized.
Colony hybridization is presumably faster and cheaper than "dot
blot" hybridization. Moreover, colony hybridization requires
specific conditions to diminish the effect of hybridization with
bacterial DNA. The marking of probes giving high sensitivity to
hybridizations should be done in order to reduce the general
background and to allow use of minimal number of bacterial
colonies. Marking of ONPs should, however, be via biotinylization,
for the benefit of easy and lasting marking in the last step of
synthesis. The sensitivity attained (Al-Hakin, A. H. and Hull, R.,
1986, Nucleic Acid Research, 14: 9965-9976) permits the use of at
least 10 times fewer bacterial colonies than standard
protocols.
In order to avoid "false positive" hybridization caused by homology
of ONP with a bacterial sequence, and in order to use short probes
such as 11-mers, which are repeated twice on average in the
bacterial chromosome, plasmid vectors that give the maximum number
of copies per cell should be used. High copy plasmid vectors, such
as pBR322, are amplified to 300-400 copies per bacterial cell when
grown in the presence of chloramphenicol. (Lin Chao, S. and Bremer,
L., 1986, Mol. Gen. Genet., 203, 150-153). Plasmids pAT and pUC
have at least twice the efficiency of multiplication (Twigg, A. J.
et al., 1987, Nature, 283, 216-218). Therefore, we can assume that
under optimal conditions even 500 copies of plasmid per bacterial
cell can be attained. The additional sequences within the chimeric
plasmids will surely cause a decrease in plasmid copies per cell,
especially in the presence of poisonous sequences. Therefore,
operations should be performed with about 200 copies of chimeric
plasmid per bacterial cell. This means that, an average, the signal
would be 100 times stronger with every 11-mer if there is a
complementary sequence on the plasmid as well. That is sufficient
difference for hybridization with bacterial DNA not even to be
registered, when a small quantity of DNA (i.e., small colonies) is
used.
Using a binomial distribution we determined how many ONPs will be
repeated more than 10 times in bacterial chromosomes due to random
order. Such ONPs will give unreliable information or cannot be used
at all if they give signals of approximate strength to signals of
all colonies.
Results obtained by using equation (1), supra, where Lf is the
length of bacterial chromosome of 4.times.10.sup.6 bp, and A the
number of different ONPs, are shown in Table 1.
This calculation assumes that all nucleotides and dinucleotides
occur in equal quantities in DNA of E. coli, which is almost
completely the case.
TABLE 1 ______________________________________ Probability of a
certain frequency of 11-mers in the genome of E. coli
______________________________________ Number of 0 2 4 6 8 10 14
repetitions Percentage of 13.5 27 9 1.2 0.086 0.004 7 .times.
10.sup.-4 11-mers Number of all -- -- -- -- 1720 80 0.14 11-mers
______________________________________
Table 1 shows that the repetition of any 11-mer greater than 13
times cannot be expected, and that the total number of those
repeated more than 10 times is about 300. Therefore, the majority
of 11-mers will have a greater than 20 times stronger signal, which
is due to the cloned DNA, than the one due to the bacterial DNA.
Naturally, a certain number of 11-mers will be very frequent in
bacterial DNA due to functional reasons, but, since bacteria do not
allow any higher degree of repetitiveness, due to their capability
to recombine, it can be expected that the number of such 11-mers
will be small. They would simply not be used for hybridization.
The problem of hybridization with bacterial DNA can also be solved
by selective prehybridization if "cold" bacterial DNA is used. By
preparing this DNA in fragments larger than 100 bp and smaller than
approximately 10,000 bp under stringent hybridization conditions
(where only the fragments with homology larger than 50 bp
hybridize), the bacterial DNA would preferentially be "covered",
since there is an insignificant chance for random homologous
sequences between bacterial and eukaryotic DNA 50 bp long, and
more, to exist.
The selective prehybridization in the sequencing method allows the
use of more probes at the same time by means of colony
hybridization. The necessary number of independent hybridizations
can thus be reduced. On the other hand, in order to know which ONP
(ONPs) permit the combination to hybridize positively, every probe
must be found in more combinations, which increases the necessary
mass of every ONP. But, if it is necessary to attain a certain
concentration of probe in the hybridization liquid for successful
and quick hybridization, and since the probe is minimally spent and
the concentration is, therefore, insignificantly reduced after the
hybridization, there is a possibility of adding a larger number of
filters into the same hybridization liquid, thereby reducing the
quantity of necessary probe(s).
By using 30 ONPs per one hybridization and by repeating one ONP in
three combinations, where none of 90 other probes is found in two
or three combinations (on account of triple the necessary mass of
every ONP), a ten-fold reduction in number of hybridizations is
achieved. On the basis of probability that a combination of a
certain number of ONPs hybridize with a fragment of genomic DNA of
a certain length, we determined the percentage of information being
lost in relation to the separate use of every ONP.
The average distance of homologous sequences for 30 ONPs of 11
nucleotides in length is approximately 130,000 bp. For sequencinq
of mammalian genomes, in order to provide more accurate reading, a
proportionally greater number of ONPs having a more frequent
homologous sequence (i.e., containing more A and T bases), would be
synthesized in order that the average distance "A" would in this
case be approximately 100,000 bp. The probability that the
combination of 30 ONPs will hybridize with a fragment of genomic
DNA having the length of Lf=5,000 bp was determined using the
equation P(Lf)=1-(1-1/A).sup.Lf. In this case, P=0.0485. The
probability that three different combinations will hybridize with
the same fragment is 1.25.times.10<Since 2 million colonies
(fragments) are hybridized, in about 250 colonies all three
combinations with one common ONP will hybridize with at least one
of their probes. We will not know whether these colonies will have
the sequence complementary to the common ONP. Since the number of
colonies which contain at least one complementary sequence ONP
common for these combinations, for mammalian genomes, is from 300
to 3,000, the number of colonies that will hybridize at the same
time with a common probe will, in the worst case, be less than 4.
For a million different ONPs a maximum of 4,000,000 information
units are rejected if we suppose that the common ONP does not
hybridize when we are not sure whether it does or does not. This
results in the loss of only 1/1,000,000 part of information
obtained from the separate hybridization with every ONP.
A greater possibility of information loss lies in rejecting the
positive hybridization when there is an ONP common for three
combinations which hybridize with the specific genomic fragment due
to a wrong determination that there is at least one ONP in every
combination which hybridizes with the specific fragment. Such
errors occur in determining the positive hybridization for every
ONP in three observed combinations, while observing whether the
other two combinations in which they are present hybridize or not.
If the other two combinations hybridize, there is a great chance
that the positive hybridization comes from the common ONP. But, if
the combinations are large, the chance that the two different
probes hybridize in one combination each is great. That would mean
that the common ONP probably does not hybridize, and so the
initially observed ONP should not be rejected as the one that does
not hybridize with the specific fragment. This probability (Pgi)
can approximately be calculated by using the equation
Pgi={[P(lf)].sup.2 .times.K}.sup.3, where K is the number of ONPs
in the combination, and P(lf) is the probability that at least one
of K ONPs hybridize on one fragment of the genomic DNA of length Lf
(equation (1), supra). The formula is valid for P(Lf).sup.2
.times.K<1. When the fragments of length Lf=5,000 bp are
sequenced, 0.1% of information is lost with K=30 ONPs, 0.5% with
K=40 ONPs, 1.32% with K=45 ONPs, 3.3% are lost with K=50 ONPs and
16% of information is lost with K=60 ONPs. We can conclude that the
reduction of necessary number of hybridizations of 10 to 15 times
can be attained with small information loss. The necessary quantity
of filters would also be 10-15 times less, such as the number of
replicas to be made out of 2 million clones.
The total number of hybridization points can also be reduced with
the use of large combinations in several steps of hybridization. On
account of 2-3 thousand additional hybridizations and two or three
rearrangements of hybridizational points, every dot could thus be
searched with about 3 to 4 times less hybridizations (i.e., so many
fewer instances of application to the filter would be
required).
Hybridization of isolated plasmid DNA would make the hybridization
method easier, but this requires the isolation of a large quantity
of plasmid DNA from many clones. The number of clones with
fragments of 5,000 bp each for the triple covering of the mammalian
genome is 2.times.10.sup.6. The necessary quantity of DNA (Mp) from
every clone is specified in the equation:
DP is the size of the chimeric plasmid in bp, DONP is the length of
ONP, Bh is the number of rehybridizations of the same filter, and
Md is the mass of DNA which can be detected in the process of
hybridization. If we take the most probable values--for DP=8,000,
DONP=11, Bh=2.times.10.sup.4, Br=10, and Md=0.1 pg, we find that it
is necessary to isolate about 0.2 mg of DNA of every chimeric
plasmid. A successful hybridization of filters hybridized with
biotinylated probe has not been developed yet. On the other hand
there are indications that it is possible to detect even 1/1000 of
pg with the biotinylated probes, Therefore, from each of
2.times.10.sup.6 clones 0.1 mg ought to be isolated.
Amplification of the whole genomic DNA can be done in about a
million portions of up to 10,000 bp, whereby the genome would be
covered more than three times. This can be achieved with the use of
a suitable mixture of oligonucleotides as primers (our patent
application Ser. No. 5724 dated Mar. 24, 1987.) With 50,000
different oligonucleotides that have about 800 repeated
complementary sequences in the unrepetitive portion of mammalian
genomes (say a 12-mer with C+G from 1-5) a million amplified
reactions with combinations of 50 primers each can be performed,
such that every primer combines with every other primer only once.
With such combinations of primers on 60 loci within the genome,
there will be on average two primers in such orientation such that
their 3' ends are facing, and are at a distance shorter than 300
bp. Fragments limited by those primers will amplify. Since their
average length is about 150 bp, the total length of the amplified
genome is about 9,000 bp. A million of such amplification reactions
replaces the plasmid or phage library of a mammalian genome. In the
amplification, the primers that enter the highly repetitive
sequences (those that repeat more than 2-3,000 times) cannot be
used, and therefore only sequencing of unrepetitive portions of the
genome would be performed. Besides, with 50,000 primers at a
frequency of 800 times per unrepetitive portion of the genome,
about 10% of the unrepetitive portion of the genome would not enter
the amplification units. With the use of 100,000 primers only, 0.1%
of the unrepetitive part of the genome would not amplify. With
100,000 primers it is necessary to perform 4 million amplification
reactions.
Using the dot blot hybridization of amplification reactions with
oligonucleotides serving as primers and newly synthesized ONPs up
to the necessary number of about 1 million, only the sequences of
amplified fragments would be read, since, with the amplification of
2.times.10.sup.4 times, every ONP with the complementary sequence
in the amplified fragment would have from 3 to 1,000 times greater
number of targets than when it hybridizes with the homologous
sequences found only within the non-amplified portion of the
genome. A signal 3 times stronger is expected for ONPs 11
nucleotides long which do not contain C or G, and 1,000 times
stronger than the 12-mers without A or T. In this analysis it is
obvious that for the sequences of regions rich with A or T the ONP
longer than 11 bp can be used (12-mer would give a signal 10 times
stronger than the background, and there is no possibility of
selective prehybridization.
After each cycle of hybridization, the technological procedure is
continued by reading of results of hybridization. Data are stored
in the memory of the computer center. Data have binary characters
(+,-) and are read from several sensitivity thresholds. Based on
these, SFs are first formed, and it is followed by mutual ordering
of SFs and subclones. At the end of processing of all the data, the
computer center determines which subclones must be treated
experimentally and what type of treatment should be applied in
order to obtain the complete sequence.
SBH is the method which minimizes experimental work at the expense
of additional computer work. The only technological requirement is
the sequence-specific hybridization of ONP. An incapability to use
up to 6% of predicted ONPs can be tolerated, with a prevention of
appearance of gaps in reading of a DNA sequence. In order to
decrease the number of falsely negative hybridizations
(unsuccessful hybridization of ONPs, since their limiting
hybridizing length is up to 11 nucleotides), and to eliminate
falsely positive ones, predicted ONPs have unspecific bases at the
ends, which are practically the only place where faults may appear.
Instead of the (N3)N8 type, (N2)N8(N1) groups of ONPs anticipated
for measuring of repetitive sequences are synthetised in the form
of the ONP group also: (N1)N.times.(N1). In the case of some basic
ONP groups which give many falsely negative matches, ONP groups of
the (N2)N8(N2) type are used, with hybridization at the temperature
characteristic for 11-mer ONPs.
Forming of internal duplexes in DNA bound to the filter is one of
the recognized structural reasons, which can produce falsely
negative matches, i.e., gaps, in reading of a sequence. This
problem can be overcome by improved binding of DNA to nylon filters
and by fragmentation of DEA prior to applying on a filter
(ultrasound, acid, endonucleases) in fragments of average size of
50 bp. A significant number of recombinant molecules will be cut
inside the duplex structure, thus preventing its formation and
making accurate hybridization possible.
Solutions upon which this method is based enable one to obtain
enough data in an entirely automated, computer-guided plant from
data in the form of binary signals; computing generates the
sequence of complex, cloned DNA fragments and/or molecules,
respectively.
* * * * *