JPH02107200A - Nucleotide sequence - Google Patents

Abstract

PURPOSE: To enable the detection of the presence of a detecting site by identifying equivalent points positioned at both ends of each of known site and the detecting site in a DNA nucleotide sequence and measuring the distance between both ends.

CONSTITUTION: The presence of a variable length site in a DNA sequence containing at least one known site (preferably a mini-satellite region) is detected by identifying equivalent points positioned on both ends of each nucleotide sequence of two or more non-identical DNA nucleotide sequences each containing a variable length site and known site and comparing the mutual distances of the equivalent points of both nucleotide sequences.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for detecting an unknown variable length locus in a DNA sequence containing at least one known locus.
The present invention relates to a method for detecting the presence of "able length focus". In particular, the method uses “informative loci” such as minisatellites or VNTR regions.
4 used for the detection of
The invention also relates to novel length variable loci and corresponding hybridization probes detected by the above method. These are used in genetic analysis methods and in the diagnosis and treatment of genetic diseases and cancer.

The possibility of using highly variable loci as informative markers for the construction of detailed linkage maps of the human genome has been investigated for several years (1) (numbers in parentheses refer to reference numbers listed below). (hereinafter the same shall apply),
The first hypervariable locus
It has recently been shown that D14S1 (2, 3), designated as D14S1 (focus), is composed of tandemly repeated short sequences (4). 5'f of insulin gene
! l (5>, c = Ha-ras 1 Shodenshi 3
’ side (6) and type ■3” side of the collagen gene (
7) minisatellites, and three minisatellites within or near the α-globin gene cluster (8 to 1
Several other hypervariable tandem repeat minisatellite loci were discovered serendipitously in human DNA, including 0). More recently, a 10-16 bp "core" sequence that is common to many human minisatellites has been identified and used to create hybridization probes that can detect a variety of minisatellites in human DNA (UK). Patent No. 2166445> (11) Polycore probe detects 60 hypervariable loci and has individual specific DNA
A fingerprint (individual-spec)
cific DNA (12), these 10-bums can be assembled into a human cosmid library (huma++ cosmiBerprint) (12).
An additional 1000 loci in the genome are detected as determined by the abundance of clones that hybridize to the polycore probe in the d 1 libraries (A, J effreys, unpublished data). These more discovered minisatellites tend to be short and do not contribute to the large and variable minisatellite populations that create DNA fingerprints.

Segregation analysis of hypervariable DNA fragments detected in human DNA fingerprints
lysis) showed that the detected loci were autosomal in origin and classified independently (11.13).

Further evidence that minisatellites are dispersed throughout the genome came from analysis of the segregation of DNA fingerprint fragments in recombinant inbred mouse strains (14).

Of the 13 mouse minisatellite loci defined, 8
The locus was mapped to the interstitial chromosomal locus of the mouse chromosome. However, the remaining five loci did not show any significant linkage with any known mouse loci, so they are presumed to be located in regions of the mouse genome lacking markers. From the above, the possibility is raised that minisatellite 1 is not scattered completely randomly in the mouse genome.

Apparent minisatellites were cloned from the human I-DNA fingerprint and locus-specific probes were generated for individual hypervariable loci with heLerozygosity of 85-99% (E
P patent application, publication number 238329) (15°16
). By screening a human cosmid library by hybridization with oligonucleotides based on known minisatellite repeat sequences, probes that detect human variable (VNTR) loci (average \70% terror accessibility) were generated. and more widely collected (17).

It has been shown that the variable loci detected by Polycore 10-B tend to be located in genomic regions known to be rich in hypervariable loci in the G band at the ends of human autosomes.
Although it is not absolute, it was found. Detailed mapping and sequence analysis of minisatellite regions detected by locus-specific probes unexpectedly revealed two closely linked minisatellite loci. These two closely linked minisatellite loci were shown to be the result of independently occurring amplification events. This means that, for example, in known minisatellite regions, abnormal differences in allele length, such as the length of acyl DNA fragments that are not due to allele length differences at known loci, are This led to the discovery that new length-variable regions can be used to detect.

One aspect of the invention is to provide a method for detecting the presence of a length variable locus in a DNA sequence comprising at least one known locus, the method and detecting, in two or more non-identical DNA nucleotide sequences each containing a known locus, an equivalent point at opposite ends of each of said nucleotide sequences, and each point between the equivalent points. The method of the present invention, which consists of detecting the presence or absence of a length-variable locus by comparing the distance between Conveniently, the method of the present invention further includes the step of analyzing the DNA nucleotide sequence by referring to the respective molecular weights, etc. is performed by convenient means such as gel electrophoresis.

As the known locus, for example, a minisatellite region, a locus with variable length, such as an informative locus (defined below) as necessary, is used.

If the known locus is a length-variable minisatellite region, the presence of a further linked length-variable locus indicates a difference in allele length that is not due to a difference in allele length in the known minisatellite region. Detected by identification.

Known loci and length-variable loci to be detected may range to any suitable distance, e.g.
Distance to is desirable.

The method of the present invention provides a new, desirable and informative method of detecting length-variable loci.

In this specification, [informative loci (inf
omaLive genej, ie focus)
” to an unrelated pre-selected], O
It is defined as a locus for which at least three different alleles can be detected in any one trial. This is expressed by stating that the locus has an allelic diversity of at least 3 (100), preferably 500 unrelated individual samples, more preferably 1000, and at least 3 in such samples. Different alleles are designated as 3 (500) and 3 (1000), respectively. By screening a sufficiently large number of whole population members, it is always possible to identify 100.5001.000 individuals with fewer than 3 alleles at the informative locus in question, so 100.50
A truly random selection of 0.1000 unrelated individual samples is required.

This vorimorphism of problematic informative loci (
The higher the locus-specific probe (polymorphisv+) is, the more useful and informative the locus-specific probe is for genetic analysis, such as in identifying paternity or individuals for forensic evaluation.

Equivalent points in the non-dentical DNA nucleotide sequence
i val entl)Oin t) are considered markers located in the region common to all sequences. The region common to all sequences can be as short as only two contiguous nucleotides, and is conveniently between 4 and 6 residues, although it is not limited to its total length. Common sites are enzymes, probes, or signaling groups.
easily identified by DNA fragments are a convenient means of providing markers, resulting in truncated ends of the DNA sequence being markers. Cleavage may be accomplished by any convenient method, but enzymatic cleavage is preferred, and more preferably restriction endonucleases are used. Any convenient restriction endonuclease can be used. Examples of individual restriction endonucleases are Nucleic
Ac1ds Re5search, 5equence
sSupple+nent, Volume+ae 16
, 19889+), r271-r313, and”
Current Protocols in M
1987-1988
F, M, Ausubel, R
, Brent, R, E, Kingston, D, D, Moore.

J, A Smith (S m1Lh), J, G,
WileyInterscience, Chapter 3, edited by Seidman and Strut+l.
It is shown in Table 3.1-1. Individual restriction endonucleases include Neo I, EcoRI, and EcoR,V. It will be readily apparent that the cleavage site should be chosen such that known sites, such as known informative sites, are not removed or disrupted.

Consequently, in a further aspect of the invention, the cleavage site defines an equivalent point and the sample fIDN.
A is cleaved to obtain the intervalvular DNA nucleotide sequence, and the presence or absence of length-variable loci is determined by the cleaved intervalvular-D
Detected by comparing the lengths of NA nucleotide sequences.

As discussed in more detail below, comparison of distances between equivalent points can identify any differences in length. It will be appreciated that if known loci are length variable, this variation must also be taken into account.

Identification of equivalent points involves routine experiments to determine whether they extend beyond the ends of the desired nucleotide sequence.
(entation) is required. If, for example, DN
If equivalent points are selected that are frequently present in the A polynucleotide sequence, then they span both ends of the DNA nucleotide sequence containing the known common region or locus and the variable length site to be identified. However, if equivalent points are chosen whose distances are too rough, in addition to the known loci common to all sequences, There may be one or more length variable regions or hypervariable loci present in the protein.

Additionally, equivalent points closer to a known common locus can be identified to more precisely localize the diversity head or locus that is most closely linked to the known locus.

DNA can be derived from any convenient source, such as genomic DNA.

DNA used in the claimed method
An example of a specific source of
erase cbainreaction: P C
R) can be used to amplify DNA nucleotide sequences for use in the methods of the invention. Amplification of the minisatellite region was performed by A.J. Sheffries (J.E.
rfreys) et al., Nucleic Acids Re
5earcl+, (1988) 16.10953-10
971. Alternatively, the sample DNA can be amplified by linear amplification, in which the sample DNA is always used to prepare a complementary copy of the sample DNA nucleotide sequence.
When the A nucleotide sequence is prepared by amplification of sample DNA, the equivalent point is
given arbitrarily by both ends of the A nucleotide sequence,
Then, cutting of the DNA is not necessary.

Consequently, in a further aspect of the method of the invention, the intervalvular-nucleotide sequences are amplified and the presence or absence of length variable loci is detected by comparing the lengths of the amplification products.

The intervalvular-DNA nucleotide sequences to be compared are e.g.
It may also be a rowed version that is included in a DNA library. Sequences to be compared are readily identified by hybridization with polynucleotides containing at least one known locus.

Therefore, in yet another aspect of the present invention, the intervalve-DN
The A nucleotide sequence was cloned.

Informative regions or loci in DNA are those that have already been identified, such as in genomic DNA, as shown in the references below.

The most convenient genomic regions for research are thought to be the regions near the ends of autosomes, with human autosomes being preferred;
Informative regions, particularly the terminal G bands of human autosomes, are particularly desirable, and are readily identified by hybridization with known polynucleotides. Suitable conditions for hybridization will be apparent to those of ordinary skill in the art. The probes are detected after hybridization using known methods, such as by incorporating into the probes a radioactive dye that is detected in a simple manner. Examples of useful 10-bums include those mentioned in the references cited above, and Lambda MS31.
Lambda MSI, Rano, DaMS8, LambdaMS32. A cloned minisatellite probe called plambda83 is mentioned.

Preferred minisatellite probes for use in the present invention include Lambda MS43 and 1) Lambda83. Furthermore, the informational locus has been applied for a British patent (patent number 2188).
323>.

It will be appreciated that if DNA from a large number of individuals is used, more information will be obtained regarding the vorimorphism of a variable length region or locus.

Fragments are produced when an equivalent point is provided by cutting the DNA. When further hybridization probes are used to identify known loci, such as rIA informative loci, probing can be performed before or after DNA cleavage, but preferably after cleavage.

The method of the invention is carried out by cutting two or more intervalvular DNA nucleotide sequences with restriction enzymes to obtain fragments containing length variable loci and known loci to be detected for each sequence; Each fragment terminates in an equivalent point such that the presence of variable loci can be detected by identifying differences in length of allele fragments that are not due to differences in allele length among known loci.

The DNA fragments used in the methods described above can be prepared by any convenient method, such as those described above. Therefore, DNA fragments each terminating in an equivalent point can be obtained by cleaving each intervalvular DNA nucleotide sequence with the same restriction endonuclease.

The regions or sites detected by the methods described above can be further analyzed by determining the DNA nucleotide sequence using methods known in the art. DNA nucleotide sequencer
A DNA nucleotide sequencer, preferably an automated DNA nucleotide sequencer, is convenient.

Yet another aspect of the invention provides new length variable DN, A regions or loci detected by the methods of the invention.

New variable regions have already been identified in genomic DNA using the method of the present invention. UK Patent Application Publication No. 218832
A linked minisatellite called Lambda M343B was detected using a DNA probe called Lambda M343 described and claimed in No. 3, and its base sequence was determined.

↓, the mentioned minisatellite region has a nucleotide sequence that can be identified by hybridization and, for example, a labeling component (IHI) el co+eponen
t) or easily identified by a nucleotide sequence with a marker component. Such nucleotide sequences may contain any number of complementary nucleotides, provided that the minisatellite region is identified by hybridization. Advantageously, the nucleotide sequence is at least 70%, 80%, 90% or 95% homologous to the corresponding minisatellite region.

As described above, in a further aspect of the present invention, the base sequence T
GGTGATGGGGCTGGACC:CGCGCG
, or a tandem repeat sequence thereof, or a sequence complementary thereto.

Advantageously, the nucleotide sequence comprises at least 10 consecutive nucleotides, preferably all the nucleotides mentioned above. It will be appreciated that tandem repeat sequences may be advantageous as well, but this is not absolutely necessary. There were 42 repeats in the isolated sequence. The sequence is linked to a sequence called MS43A which is described and claimed in UK Patent Application No. 8706401.

In addition, polymorphic regions (po
lymorphic region) was identified using a DNA probe called p-lambda g3, which is described and claimed in UK Patent Application Publication No. 2188323. New highly diverse DNA minisatellites are closest to the p lambda g3 locus, NcoI and PvuIr.
It is characterized by being located between restriction endonuclease cleavage sites. The sequence of this region or locus was determined by methods known in the art, such as those described above.

In a further aspect of the invention, nucleotide sequences are provided by which the new loci described above can be identified by hybridization.

Further length variable polymorphic regions were identified using a DNA probe (unpublished), hereinafter referred to as pMS228. Using the method of the present invention, minisatellites consisting of this length-variable polymorphic region can be prepared as follows:
It was found to contain two linked minisatellites, designated pMS 228 A and pMS 228 B. A further aspect of the invention includes a nucleotide sequence by which one of the above-mentioned new loci can be identified by hybridization.

As described above, a further feature of the present invention is that the sequence GGGGA
GGGAGGGA(:GCTGTACTGAGGTTT
T^^T^ΔTTATT to AGC^GTGACC(:T
l;TCTCA (: [:^^^ The purpose is to provide a nucleotide consisting of at least 8 consecutive nucleotides selected from GAGAT, its tandem repeat sequence, or a sequence complementary thereto. The nucleotide sequence must be at least 10 consecutive nucleotides. Advantageously, it consists of nucleotides such as, preferably all the nucleotides mentioned above.

As described above, a further feature of the present invention is that the sequence GGG has G
ACAGGGC is provided with nucleotides consisting of at least 8 consecutive nucleotides selected from RG^, its tandem repeat sequence, or a sequence complementary thereto. Advantageously, the nucleotide sequence consists of at least 10 consecutive nucleotides, preferably all the nucleotides mentioned above.

Yet another aspect of the invention provides a DNA probe comprising a nucleotide sequence according to any of the above aspects of the invention together with a labeling or marker moiety.

Examples of the invention are described below without limitation with respect to the detailed description below.

(Materials and methods) Probe mark.

^M S 31 , λMSI, λMS8. λMS32゜＾MS43 and pλg3 (15,16) 1 block 3A
The I insert was digested with the restriction endonuclease ΔMo-2Ha
By digesting with one or more of e[, Hin f I, clones (D
NA (f Iankin8D NA ) was removed. Minisatellite DNA fragments were purified by gel electrophoresis (18) and 0.3 nl Aole of I-dA.
TP, I (-dCTP and codTTP 2 to 8 ×
Oligo-labeled in the presence of 10' cpm/μg (
19), the multi-loci (mult
ilocus) M 13 recombinant probes 336 and 33.
Minisatellites derived from 15 are digested with Bayu H1 and Fuhada I, or BamHI and EcoRI, respectively, and converted to M1.
3 Excise from RP DNA and 1H as described above.
-(- labeled.

In 5 itu hybridization MS31.
^MSL, λMS8. λMS32゜＾MS43 and p
The labeled probe derived from λg3 was purified as previously described (21
, 22), 100 μg, /+ oIBrdUrd <20.21) -240 μg/J of low molecular weight denatured salmon DNA was hybridized during metaphase of normal human lymphocytes cultured. Multi-locus probe 33
．． 6 and 33.15 were similarly hybridized without competing NA. slide glass to ilford 2
Nuclear research emulsion (l1fordK2 Nuclear
Re5eareb Emulsion) at 4°C.
Soaked for ~14 days, followed by Vexto 3.3258/UV/
Giemsa method (tbe Hoechst 33258/
A G band was formed using UV/Giemsa PA method (20).

``Wataru'' ΣN Noyo DNA was obtained from white blood cells of an unrelated northern European individual (12); other DNA was obtained from Ce'ntre du
Obtained from Polymorphis+ae Human.

Genomic DNA was digested with restriction endonucleases and electrophoresed as previously described (13
), the DNA was converted into Hyboncl-N (Amershan International: Amershan + I nLer
national) and Church (Churc).
h) and 33p-labeled DNA'70-b by Gibert (23) were hybridized.

A total of 22 days of human genomic DNA digested with au3AI was size selected and ligated to λL47.1, and the resulting library was screened with core probes 33.6 and 33.15. Positively hybridized clones (λMS series) are
When used as a probe of genomic DNA, it does not recognize highly diverse loci. A similar method was used for the isolation of λMS22S and then the 5au3AI insert (inser
t) was subcloned into the BamH1 site of pU C1,3 to obtain pMS228. Further analysis of pMS228 is described in “Results” and summarized in Table 3.
5itu hybridization was performed as described (50). DNA sequencing was performed using Klenow fragment, modified T7 polymerase (52) or Ther+nus aquatica (Ther+nus aquaticus).
The dideoxy chain growth termination reaction method (dideoxy c
hain termination meth
od) (51).

(Results) Detecting one highly diverse locus each, p
λg3. λMSI, AMS8. λMS31. λMS32
．． Six cloned minisatellites called MS43 (Table 1) <15.16> were hybridized in 5 in situ to human metaphase chromosomes.

We have already shown that these blobs are very high in Southern blot analysis, probably because both the 70-b and the target locus are tandem repeats.
As if you've been waiting! The signals of the n5 itu hybridizations were strong and dark, and many of the metaphases showed one or more silver grains in the associated loci-bands (Fig. 1), resulting in relatively few localized cells being analyzed. (Table 1).

Figure 2 shows the distribution of silver particles after t-''±u hybridization with each of the 611 minisatellite probes. Each probe detects one prominent locus and these loci are located on chromosome 1, 5.7.12.
The above regional localization (Table 1) completely matched the chromosomal distribution obtained from somatic cell hybrid analysis (16). 10-in 5it using λPvI S 32
U-hybridization analysis showed a relatively high randomly distributed background with clear localization to 1q42-43.

The distribution of silver particles after hybridization with λMS8 was found to include 5 q35-qter major loci, plus 12
A second minor peak was shown at q24.3-qter. The second peak may represent another locus with a sequence similar to the D5343 locus recognized by λMS8.

Similar to the second locus detected in ^MS8, 4 of the 6 minisatellite loci are localized to the terminal 0 body (Table 1), and the remaining minisatellites λM Sl and ^MS3
2 are both localized near the end of chromosome 1, but there is a statistically significant, although not perfect, strong tendency to localize in the distal G band rather than at the end. - Metaphase cells with about 400 G bands per double wood getum were used for these analyses. If DNA were evenly distributed on chromosomes, 11% of the genome, and consequently 11 randomly distributed minisatellites, should be present in the terminal G band. Therefore, it is significant that 4 out of 6 minisatellites are localized in the terminal G band (P=0.002 for random distribution).

This non-random distribution of minisatellites is consistent with multilocus DNA fingerprint probes 33,6 and 33.15 that detect 1000 Ji positions in the human genome.
Supported by 5 itu hybridization. compared to the 11% expected with a random particle distribution.
26-32% of all chromosomal silver particles were localized in the terminal G band (Table 2); however, the efficiency of detecting different minisatellites with these probes varied from locus to locus; It is not yet possible to predict the proportion of all minisatellites and the proportion of hypervariable loci without localization near the ends.

For mu DNA, pλg3. λMS8. Similar experiments with λMS31° and λM843 found no evidence for "smeared" bands indicating a physically close linkage with telomeres (data not shown); The minisatellites are not telomeric and are located at least 12 times from the telomere.
It is suggested that they are located kb apart. The maximum distance from the telomere is 8 mb, which is the approximate length of the DNA in the terminal G pot.

Telomeres on the short arms of human X and Y chromosomes were identified with probe 291C, which defines the DXYS14 locus, which maps very close to telomeres. Appropriate restriction fragments containing DXYS14 and telomeres showed 14 kb diversity due to heterogeneity in telomere length among individuals and showed “smeared” hybridization bands on Southern blots (25), 14. The tendency of minisatellites to localize in the genome-terminal G band cut with different restriction endonucleases suggests that these regions are rich in hypervariable loci. Two lines of evidence support this possibility.

As previously mentioned, λMS43 detects a secondary variable locus in addition to the primary hypervariable locus (16); pedigree studies have shown that these two loci are closely linked. (unpublished data), detailed mapping and sequence analysis of the HiI-S skin 3A DNA fragment cloned into λMS43 revealed that a second minisatellite MS
-'13 B is 1 from the main minisatellite M543A
．． This second minisatellite, found to be located at 0 kb (Fig. 3A), is highly sensitive to both the "core" sequence and the DNA fingerprint probe 33.6 used to identify λM343. It consists of 43 repeats of 24 bp of G-rich sequence containing similar regions.

In contrast, a major G-rich minisatellite with a repeat unit length of 45 bp has little sequence similarity to the core or 33.6
rarity) (1G), from the subsequent experiment, ^M 343 has a probe of 33.6 teMS-43
A Tehana (due to the detection of MS 43 B)
) (unpublished data), there is no significant similarity between these two minisatellites, which are G-rich and located in opposite orientations with respect to NAffl. Although these two minisatellites are closely linked, the above results indicate that they appear to have arisen through independent amplification, as described in this specification (no changes have been made to the content).

The second minisatellite M S 4.3 B serves as a locus-specific probe (Figure 3B). The detected locus has a repeat copy number of 524-50 with alleles of at least 7 different lengths and an associated heterozygosity of 35% (Figure 3C). In contrast, the major minisatellite 1- detected at M34.3 has more than 20 different length alleles and shows 96% heterozygosity (16).
.

In order to extend the search for chained minisatellites, each minisatellite that was
Human F-D cut with 1 different restriction endonucleases
Hybridized with NA. In cases where there is length heterozygosity at the z locus, there are abnormalities in the length of allele fragments that are not caused by differences in allele length in the minisatellites detected for P I hoop. This is revealed as a significant difference. This approach allows us to identify 1) a second highly variable length polymorphic region (polymorphbic) in the vicinity of the hypervariable locus detected in λg3;
r+4ion) was found (Fig. 4A). λ
The restriction endonuclease that cuts near g3 is l) λ
Allelic fragments were produced that displayed length differences that were entirely due to the allelic diversity of g3. On the other hand, restriction endonuclease Nco ■.

Cleavage with EcoRl or EcoRV produced an abnormally sized Al/R fragment, suggesting the existence of a second variable region that is nested from pλg3. genomic mappinF3
), this second variable region extends from pλg3 to 2kbQ
It was shown that NcoI-PvulI was located in the sequence (Fig. 1B). Among the 21 alleles extracted from individuals in the CE P H tree, this interval showed a large number of alleles with a diversity of lengths ranging from 4 to 36 kl. pλ
There was no significant correlation between the length of the g3 allele and the length of the second linked hypervariable locus (Figure 4C).
).

Subsequent studies showed that the two loci were detected with high stringency by pMS228.

Pedigree analysis showed that these loci are closely linked. Restriction enzyme map of cloned DNA,
Subsequently, the base sequence analysis confirmed that 228A and 228
The presence of two different tandem repeat regions, designated B (Fig. 4), was demonstrated. A subclone derived from 0M8228 (su
Be! 228A is larger, from human DN (reprobinH) roving (reprobinH) by one).
It was shown to detect strongly hybridizing loci and 228B to detect smaller, weakly hybridizing alleles.

These minisatellites and eyes+5 iju hybridization mapped them to 17pl 3-pter. pMS
Table 1 summarizes the nature of the loci detected by M.51 and pMS228, as well as by other minisatellites described in this report.

Both minisatellites in pM 322 B are 80%
or more heterozygosity. minizaterai l-228
In B (Fig. 5, Table 3), high heterozygosity (8
5%) and a limited Al/R size range (0,6-5.
Polymerase chain reaction (polymerasecha
This is extremely useful for minisatellite analysis (in reaction). In general, the most diverse loci have a wide range of allele sizes such that many alleles exceed the maximum size currently amplifiable by this method, resulting in incomplete amplification products, and In contrast, the locus detected at 228B has 48
As a result of investigating unrelated individuals, 95% of alleles are 2
kb, and even the largest one (5.5 kb) was within the amplifiable range (J, Armou
r) and A, J effreys.
, unpublished), we showed that amplification of a single locus yields complete and useful information. 6 of adjacent arrays
．． Within 5 kb, there are 7 distributed repeat elements (4 Δ±
Carving stone, one Ll-(Kpn), LT of retrovirus
Although there is some partial diversity in human DNA (R and recently discovered short interspersed repeat sequences), human hawkins are expected to exist every 5 to 6 kb and Ll to exist every 3 Q kb in human DNA. be done. In this way, the high frequency of Alu and L1 elements suggests that there are an excessive number of anti-Japanese sequences scattered near the minisatellites. Interestingly, human tissue plasminogen stimulating factor (1+human tissue plasminogen
inogen activator:t-PA
) Interspersed repeat sequences have been shown to be present at a similarly high frequency in the non-coding DNA of genes, including 28 complete or partial elements, partial L1 elements,
and a substantial block of 51113 tandem repeats (5701 month) were found in 36.6 kb.

Minisatellites λMS8 and λMS32 near the end.
Similar experiments using interstitial probes λMSL and λMS31 found no evidence of restriction fragment length heterozygosity in the DNA flanking each minisatellite in a single individual examined.

The location of four of the six hypervariable minisatellites in the distal G band of human autosomes, as revealed by in 5 in situ hybridization, is statistically significantly biased from a random distribution. in 5 at multilocus probes 33.6 and 33.15.
2 at the telomere after iLu hybridization.
This is also indicated by the concentration of 6-32% silver particles. The exact proportion of hypervariable minisatellites that map to the terminal 11-proximal region is unknown, and their physical location relative to telomeres has not yet been determined.

A hypervariable locus that maps to the terminal G band is also present in Ike, c-11a-rasl 3 located at 11p15.5.
'Hypervariable region (HV R) (6), cross-hybridizes with 10q26 (cross-l+ybrid
iziB) Mini satellite VTR4, 1 (30), 11
q15.5 insulin 5' [-(V R(5)
, the locus detected by the 7q3G probe CRI 5194 (31), and the immunoglobulin heavy chain J H5' HV R located at 14432.3 (32). Similarly, the only hypervariable locus identified within a random collection of chromosome 5 DNA11I'r fragments also mapped to the G band at the end of the short arm (33,34). Furthermore, a preliminary linkage map of the human genome has recently shown that a remarkable proportion (11/28) of loci with 70% or more heterozygosity exist in the terminal 5% of the autosomal linkage map (35 ), L However, the cytogenetic location of most of these hypervariable loci has not been determined. Minisatellites tend to be located in the terminal part, but there are also examples of them being located in the middle part, DIS7 and D 138 (present invention, T(ii), 14q32.1-3).
2.2's old 4S1 (3), α-globio gene crystal located at 16p13.1 by detailed tR mapping.
three 1-1 VRs (36) in cluster)
, and 7J 12q13.1-14.3 AT-rich type ■ collagen 3°HV R (7) are examples. Relative to humans, at least 8 of the 13 hypervariable minisatellite loci detected in the mouse DNA fingerprint map
rsLitial 1oCation) (14). It is not yet clear whether this represents a significant difference in minisatellite distribution between the two species.

Not only do human minisatellites tend to be located in the distal G band, but these regions also tend to have minisatellites forming clascous. Minisatellite clone λM343 contains two closely linked but distinct minisatellites that are likely the products of independently occurring amplification events. Similarly, the hypervariable locus 1fiD 7 S 22 to 2kl+ detected in 1]λg3
A second highly variable region is located at. This clustering of autosomal minisatellites near the ends of human and mouse sex chromosomes (
It reminds me of an autosomal pair region. This region also contains a high density of hypervariable DNA fragments (37-3), at least some of which are tandem repeat minisatellites (38), one of which, DXYS14, is found in close proximity to telomeres. It is located (25). This suggests that the structures near the ends of autosomes and sex chromosomes are somewhat similar.

It has been proposed that a core sequence common to many GC-rich minisatellites is involved in causing non-homologous crossover at hypervariable loci (+1>, and this hypothesis Meiotic recombination hotspot sites in the mouse Eβ M HC gene (meioticreco+lIb1
This is supported by the presence of core-related sequences at or near natin botspoL (4
0). Therefore, in human male meiosis, recombination nodes associated with chiasma (41, 42) and the synaptone+nal complex
It is interesting that the distribution of both of these genes (43) tends to be located near the distal regions of human autosomes. More direct evidence that recombination is more likely to occur in regions near the ends of human sex chromosomes during meiosis in men, but not in women, lies in the pseudoautosomal regions of human sex chromosomes.
(37), (45,46) and tentative evidence in linkage map expansion of the termini of human chromosomes 10 and 21 (35). There is. In humans, autosomal terminal linkage map expansions are common in males (35°47), and are likely similar to those found in pseudoautosomal pairing regions of sex chromosomes. This is thought to reflect the difference in the frequency of terminal recombination events during female meiosis. Hypervariable minisatellites exhibit very high mutation rates for new length alleles (48), consistent with the theory that minisatellites are hot spots for recombination (11). Therefore, attempts have been made to link the tendency of recombination in regions near the ends of human chromosomes to the high density of minisatellites in these regions 7). However, highly unstable minisatellites are not necessarily located in the terminal G band, and in fact, two of the most unstable human minisatellites identified, DIS7 and DI
S8 (・18.1. Pertyl and A
, Jef r reys (unpublished data) are both located in the interstitial region. The relationship between minisatellites and recombination near the terminal rt is still unclear, suggesting that minisatellites near the terminal are not directly involved in recombination and/or pairing in these regions, but instead It is also possible that they arose as a byproduct of selectively promoted recombination.

Highly informative and regionally localized marker loci, such as those described herein, provide essential landmark loci for the generation of accurate human linkage maps associated with cytogenetic maps. .

Furthermore, the location of many minisatellites near the ends suggests that terminal map expansion should not lead to marker-deficient regions in the human linkage map, as seen in the qyu, reciprocal ±2L noe (44). It will be confirmed. Terminal hypervariable markers are also ideally suited for somatic cell recombination, where the marker is deleted in B tumors, particularly where homozygosity of all markers adjacent to the recombination site occurs. (4)
9). It remains to be seen whether there are enough highly informative interstitial loci to make the creation of a high-resolution mini-synapsis of the human genome a viable proposition.

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, No72e Multi-Locus DNA Fingerprint Pro,
-Distribution of silver particles on one human stain after in situ hybridization using probes Control hybridizations with probes showed that 15% background particles (bac
kground grain) L was not shown (
(data not yet published).

Number of cells analyzed + 4536 Number of silver particles on the chromosome 1191 1026 Number of silver particles on the terminal G band 309 332 Silver particles at the terminal $
26 32! Multilocus probes hybridized in the presence of competing river herring DNA showed a decrease in hybridization signal. (24,1
, Patel, R. Kelly
, O, A, J, C' Effrey (J effrey
s), unpublished data). As a result, all competing DNs
A is in s i with 3315 and 336 probes
omitted from tu hybridization.

Ten slides from two individuals were analyzed for each probe. For comparison, Figures 1 to 5 will be explained in detail below the usual single locus.

oil ↓”−λM S 43 and λMS3] for n
Two cells in metaphase showing silver particles after 5 itu hybridization. The arrows indicate λM843. for D12SIL, respectively. 1.2q24.3-qtcr and 7p22- after in 5 in situ hybridization of λMS32 to D7S21
IT Ler silver particles are shown.

First figure-11133-1135, 1q42-q43,
7p22-pLer, 7q36qter, 12q24.
Probes λM S L , λMS32° λMS located at 3-qjer and 5q35-qter, respectively
31. pλ83. λMS 113 and λMS8
His1 of the silver particle distribution on the human chromosome that formed the G band after in s it u hybridization with
~Gram Sound, -Moon King Hi■・Mini Sanarai 1~Clone λM 3
43 contains two variable minisatellites. A.

8.3kb 5au3A inserter 1- on λM 343
(16) was subcloned into the Ba+nt11 site of pUcl, 3 (26), and detailed restriction enzyme maps were constructed and analyzed: minisatellites (black squares) and Alu
, J digestion site (a) is shown. Second mini satellite l-M
A 1.5 kb Alul fragment containing S-=1313 was subcloned into the S+na site of M13+np1.927), and the minisatellite repeat unit was determined by dideoxynucleotide sequencing (28,29>. Variable minisatellite l-M
The S43A repeat sequence was previously reported (46
). B, 32p label that sounds MS43B A Iu I U
The r fragment acts as a locus-specific probe when hybridized to the AIu gene of human I-DNA. The minisatellite loci detected were diverse among the eight unrelated individuals tested. A northern European minisatellite from two unrelated individuals.
- The density distribution of M S 43 F3 was determined.

A second highly variable locus is located next to the hypervariable locus D7S22 detected in mpλg3 iP). A. Individual 136201. CI of (1) and 1.36202(2)? , PH-derived DNA sample I-(inf I
(l(>, P vu IT(P), Bclr(B)
, NcoI (N), Alui was cut with EcoRV (E), electrophoresed on 0,6z agarose, and Southern blot hybridization with minisatellite pλg3 was performed. The allele fragment (0,0) in each digestion product was traced and identified as segregating in the progeny of 136201 and 136202 (data not published)
. The difference in allele length in the P and B digestion products is consistent with the difference in size when cleaved with H, which cuts near the minisatellite D7S22 (pλg3), and is due to the allelic diversity of D7S22. Be careful 6On the contrary,
The increase in allele length of N and E digestion products varies from allele to allele, suggesting the presence of a second length polymorphic region contained within these DNA fragments. B, P, B
.

1) λg3D detected DNA1ff in N, E, R (ratio R,l) restriction digest products; haplotypic (1-1aplotypic) genome restriction enzyme map of four alleles of 136201 and 136202 determined from fragments. The second diversity locus (・−/−/′−) is pλ
It is located between P and N, 2 kb from the g3 minisatellite (black square).

C, Relationship between the size of the second variable region locus defined by the length between P-N and the length of the pλg3 minisatellite determined for 21 randomly encountered Caucasian habrotypes.

Fig. - Analysis of pMS228. (A) DNA from a certain household group (F = father, M = mother, S = son, D = daughter) digested in country A and analyzed using DMA228 as a probe.
, some alleles hybridized strongly and some hybridized weakly; strong and weak alleles that segregated together as linked pairs in children are shown in parentheses within parents. (B) 228A and CEPH
48 unrelated individuals of consanguineous origin (Δlul of 228B>
Allele length distribution. In the histogram, the allele is 0.25k
b; each size step contains a large number of analyzable alleles, and the number of alleles at each locus is much larger than the number of steps shown. (C) Structure of plasmid clone pMS228. The region indicated by a box indicates the minisatellite part, and the area below it indicates the corresponding repeating unit sequence. At high stringency 2i hybridization of human DNA, 228A detects a more strongly hybridizing band and 228B detects a weaker band.

R means A or G.

[Brief explanation of drawings]

Figure 1 shows n 5 for λMS43 and λMS31.
FIG. 2 is a diagram depicting two cells in metaphase of cell division showing the location of silver particles after iLu hybridization. Figure 2 shows each probe! II is a histogram of the distribution of silver particles on a human chromosome forming a G band after 5iLu hybridization. Figure 3A shows minisatellites MS43A and MS4
．． Figure 3B is a diagram showing the positional relationship of minisatellite MS43Bl)Crg, and Figure 3C is a diagram showing that minisatellite MS43Bl)Crg acts as a position-specific probe. It is a graph showing the relationship between the number of repeats and the frequency of seven different alleles constituting a locus. FIG. 4A is a diagram showing the results of Southern blot hybridization between a DNA fragment derived from CEPH and minisatellite pAg3, and FIG. 4B is a haplotypic genome restriction enzyme map of one allele. Figure 4 C is P
FIG. 12 is a graph showing the synchronization of the size of the second variable region locus defined by the length between -N and the length of 1) λg3 minisatellite determined for hablotybes. Figure 5A is a diagram showing the results of digesting DNA obtained from a certain family group with AluI and analyzing it using DMA228 as a probe, and Figure 5B is a diagram showing the results of DNA obtained from a certain family group being digested with AluI and analyzed using DMA228 as a probe.
This is a histogram showing the J allele size distribution, and the fifth
Figure C shows the construction of plasmid clone 1) MS228. (3 others) Procedural amendment Written by the Commissioner of the Japan Patent Office Yoshi 1) Takeshi Moon1, Indication of the case 1999 Patent Application No. 1492712, Name of the invention Nucleotide Sequence3, Person making the amendment Relationship to the case Patent Applicant Address 4, Agent (Attachment) The scope of claims is amended as follows. j2. [Vf Claims] 1. A method for detecting the presence of a variable length locus in a DNA sequence containing at least one known locus, the method comprising: a variable length locus to be detected; two or more intervalves each containing a sexual locus and a known locus - I'1l)
Identifying the equivalent points at both ends of each nucleotide sequence in the NA nucleonate sequence, and detecting the presence or absence of a quality variable locus by comparing the distances between the respective equivalent points. The detection method consists of: 2. The method according to claim 1, wherein the known locus is a minisatellite region. 3. The method according to claim 1 or 2, wherein the length-variable locus is an informative locus. 4. The method according to any one of items 1 to 3, wherein the intervalvular DNA nucleotide sequence is cloned. 5. The intervalvular DNA nucleotide sequence is amplified, and the presence or absence of a length variable locus is detected by comparing the lengths of the amplified products, according to any one of claims 1 to 54. Method described. 6. The equivalent point is defined by the cleavage site, the intervalvular DNA nucleotide sequence is obtained by cleaving the sample DNA, and the length variable is determined by comparing the lengths of the cleaved intervalvular DNA nucleotide sequences. The method according to any one of Items 1 to 3 of Patent No. 2i'l, which detects the presence or absence of a locus. 7. A nucleotide sequence capable of identifying, by hybridization, a DNA region or locus detected by the method according to any one of claims m1 to FtSG. 8. Claims containing nucleotides that are 70% or more complementary to the DNA region or locus [11i No. 7]
Nucleotide sequences as described in Section. 9. DNA minisatellite pMS228A% DNA minisatellite pMS 228B, DNA minisatellite)
MS43B, and minisatellite 1) Nc, the restriction endonuclease digestion site closest to the λg3 locus.
A nucleotide sequence by which one of the group consisting of DNA minisatellites characterized by being located between oI and Pvul[ can be identified by hybridization. 10, Sequence G(:GGAtl:(:GA(C:GAGGCTGTA
CT(:AGGTTTTAATAATTATT(:AG
CAGTGACC[;TGTCTCA(:(:A to A(
: A nucleotide sequence consisting of at least 8 consecutive nucleotides selected from AGAT or its tandem repeat sequence or a sequence complementary thereto. 11. A nucleotide sequence containing at least 8 consecutive nucleotides selected from the sequence G(:CG(:ACAGGGC(:ftGA) in which R represents A or G. 12. The sequence T contains GT(:AT(:GGCTGGA)
A nucleotide sequence consisting of at least 8 consecutive nucleotides selected from C to G to GGCG or a tandem repeat sequence thereof or a sequence complementary thereto. 13. A polynucleotide probe comprising C) the nucleotide sequence according to any one of claims 7 to 12, which has a labeling component or a marker component. 14. The nucleotide sequence according to any one of claims 7 to 12 or claim Pt51
Genetic disease or cancer treatment characterized by using the probe described in Section 3
- This is the place. 15. vf Use of the nucleotide sequences according to claims 7 to 12 or the probe according to claim 13 in genetic analysis. j and above Written amendment (method) 1. Indication of the case 1999 Patent Application No. 149271 2. Name of the invention Nucleotide Sequence 3. Relationship with the case of the person making the amendment Name of the address on the patent issuer name Imperial Chemical Co., Ltd. Industries PLC 4, agent address: Shin-Otemachi Building, 2-2-1 Otemachi, Chiyoda-ku, Tokyo 206-ku 5, amended order dated September 26, 1999 (
Shipping port) 6° correction against Australia

Claims (1)

[Claims] 1. A method for detecting the presence of a variable length locus in a DNA sequence containing at least one known locus, the method comprising: a variable length locus to be detected; Identifying, in two or more non-identical DNA nucleotide sequences, each containing a known locus, the equivalent points at each end of each nucleotide sequence, and comparing the distances between the respective equivalent points. The detection method comprises detecting the presence or absence of a length-variable locus. 2. The method according to claim 1, wherein the known locus is a minisatellite region. 3. The method according to claim 1 or 2, wherein the length-variable locus is an informative locus. 4. The method according to any one of claims 1 to 3, wherein the non-identical DNA nucleotide sequences are cloned. 5. The method according to any one of claims 1 to 4, wherein the non-identical DNA nucleotide sequences are amplified and the presence or absence of a length variable locus is detected by comparing the lengths of the amplified products. the method of. 6. The equivalent point is defined by a cleavage site, the non-identical DNA nucleotide sequences are obtained by cutting the sample DNA, and the length variable locus is determined by comparing the lengths of the cleaved non-identical DNA nucleotide sequences. Claims 1 to 3 detect the presence or absence of
The method according to any of paragraph 3. 7. D detected by the method according to any one of claims 1 to 6 by hybridization
A nucleotide sequence that can identify an NA region or locus. 8. The nucleotide sequence according to claim 7, which contains nucleotides that are 70% or more complementary to the DNA region or locus. 9. DNA minisatellite pMS228A, DNA minisatellite pMS228B, DNA minisatellite MS
43B, and the restriction endonuclease digestion site closest to the minisatellite pλg3 locus, ￥Nco￥
A nucleotide sequence that allows one of the group consisting of DNA minisatellites to be identified by hybridization, characterized in that it is located between I and \Pvu\II. 10. A nucleotide sequence consisting of at least 8 consecutive nucleotides selected from a sequence [there is a gene sequence] or its tandem repeat sequence or its complementary sequence. 11. A nucleotide sequence containing at least 8 consecutive nucleotides selected from a sequence where R represents A or G [there is a gene sequence]. 12. A nucleotide sequence consisting of at least 8 consecutive nucleotides selected from a sequence [there is a gene sequence] or its tandem repeat sequence or its complementary sequence. 13. A polynucleotide probe consisting of the nucleotide sequence according to any one of claims 7 to 12, which has a labeling component or a marker component. 14. Detection of genes that cause genetic diseases or cancer, characterized by using the nucleotide sequence according to any one of claims 7 to 12 or the probe according to claim 13 Method. 15. Use of the nucleotide sequences according to claims 7 to 12 or the probe according to claim 13 in genetic analysis.