JPS62501467A - polynucleotide probe - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 1,21F and r condyle This invention provides a useful tool for probing human or animal genomes (probiB). polynucleotides that can be labeled to serve as probes, and This invention relates to a method for immobilizing genomic DNA using lobes. This fixing method is an example of For example, it is useful in paternity and maternal lineage testing, forensic medicine, and the diagnosis of genetic diseases and cancer. be.

2. CM introducing “Kitsune Maze” The main conventional method for fixing genetic differences in genomic DNA is restriction fragment length. restriction fragment length poly morphismS; RFLP). for example, J, F., Gusella et al., Nature 306.234-238 (1983 ) and W. According to Cavanee et al., Nature 305.779-7 (1984). See Analysis of Retinoblastoma Predisposition.

Most RFLPs involve small changes in DNA, usually restriction endonucleases. resulting from base substitutions that create or destroy enzyme cleavage sites. Average heterogeneity of human DNA The zygosity (mean hetero-zygos ity) is low (per base pair (approximately 0.001), so restriction endonucleases RFLP will only be detected infrequently, and even when it is detected, most R FLP depends on allele frequency, which never exceeds 50% and is usually very low. Dimorphic with hybridity (diIIIrphic) determined by (presence or absence of a donuclease cleavage site). As a result, like this All RFLPs are included in the pedigree analysis whenever the definitive individual is homozygous. It is homogeneous.

Genetic analysis depends on the availability of probes for hypervariable regions that exhibit multiple allelic variation. more, and correspondingly higher heterozygosity (he terozygos i ty ) could be considerably simplified.

A first such domain is A.R. Wyman et al., Proc.Nat.Acad. , Sci.

USA? ? , 6754-6758 (1980) (coincidentally, h) Isolated from a library of random segments of DNA. The polymorphism of this locus Structural structure of multiallelic variation The basis is still unknown. Later, and also coincidentally, several other highly variable regions is the human insulin gene (G, J, Be11, etc., Nature 29531- 35 (1982)), zeta globin gene (N, J, Proudfo ot et al., Ce11 3L 553-563 (1982), and S, E, Y, □odbourn, etc., Proc, Nato Acad, Sci, USA, 8 0.5002-5026 (1983)), and C-■a-ras-1 oncogenetic gene Child (1), J. Capon et al., Nature 302.33-37 (1983 ) was found in the vicinity of ). In each case, the variable region is a short sequence (“minisatellite”) consists of tandem repetitions of ``light'') and polymorphism. ) is - mitotic or meiotic non-excha caused by DNA slippage during replication. Based on allelic differences in repeat number. The difference in length of the resulting minisatellites is due to the repetition It can be detected using restriction endonucleases that do not cleave the unit.

The present inventor and his co-researchers have already discovered that the intron of the human myoglobin gene describe a short minisatellite comprising four tandem repeats of a 33 bp sequence of (P. Weller et al., EMBOJ, Yu, 439-446 (1984) ). This 33 bp repeat has been previously characterized in other human-minisatellite species described above. It was noted that it showed weak sequence similarity to This sentence is mi It is suggested that the nisatellite region is generated by transposition. Established. 33 bp repeats of the human myoglobin gene are transposable able), this is considered to be multi-allelic variability based on the difference in the number of repeats. Provides probes for frequently related Kundem repeat regions of the human genome The human genome DN8 is a tandem sequence of 33 bp sequences from the myoglobin gene. probing with a DNA probe comprising Ambitious phase Differences occur in several different regions in the genomic DNA of three individuals (father, mother, and daughter) This difference occurred in the size of larger fragments (2–6 kb). Ru. The data are stable due to differences in the length of more than one minisatellite region. consistent with the reported variability.

2 lessons, 1 plate per month -5 studies using myoglobin gene 33bρ-repeat probes. game in a way that much more effectively displays variability in minisatellites or hypervariable regions. It was shown that it is possible to search for genomic DNA.

This invention shows that many minisatellites in human or animal genomic DNA are different minisatellites. Discovery that satellites contain a 9M region with a high degree of homology It is based on This common core region is short, approximately 16 base pairs. Nowadays, there are few Its essential composition consists of a core sequence of nucleotides repeated at least three times in tandem. The probe it has as a component can probe many different minisatellite regions in genomic DNA. , and by referring to the differences in their DNA in these regions, individuals can This may help to identify or detect with enough precision that it can be identified. was found. These conventional probes are more effective than those provided by RFLP. Fingerprints that lead to a good degree of identification but are essentially specific to an individual erprint). Notably, more than one mini satay By reference to the light region or hypervariable locus, the core probe of this invention is D It has been found that NA can be fingerprinted, and an unusual degree of involuntary It is this discovery that adds clarity to inventiveness, which is fundamental to scientific novelty. This makes it an important invention.

From the knowledge of the core sequence, minisatellite regions from various loci in the genome and DNA probes with hybridizing properties can be produced. deer However, mere recognition or identification of a specific core sequence may not, in itself, be a useful protein. Insufficient for the manufacture of robes. Contains core region Kundem repeats It is also necessary to produce polynucleotides or derivatives thereof. Such a professional The probe can be isolated as a minisatellite from human or animal DNA. , or alternatively can be created by synthetic techniques. Hybrid with good results It is also important to achieve additional constraints for performing dizization. These are acceptable consensus - degree of homology with the core and within the repeat unit as a whole This includes knowledge of acceptable lengths of non-core DNA.

Thus, the invention employs the following realizations and discoveries: (1) Any one core configuration Knowledge of columns can be used in this method. This further includes the recognition of: (2) By studying different hypervariable loci in the genome, we can that different core sequences can be found with a sensus; (3) Accumulation of more DNA probes that would recognize variable vectors what can be done; (4) Certain gene classifications are preferred by some probes over others. that can be achieved in the result; (5) more than one professional in any one classification; (6) the use of successful probes amplifies the probability of identification; A generation of further research has led to simpler and more successful probes, e.g. can be manufactured by

Thus, according to the first aspect of the invention, the polymorphous minisatellite - length-specific A method of producing a polynucleotide having binding properties is provided, the method comprising: (i) Restricted hybridization to other polymorphic DNA' pM regions identify natural tandem repeat sequences in DNA that can perform (ii) the natural consortium of said repetitive sequences putatively responsible for such binding; identify the sus-core sequence and (iii) lower the genome than the natural repeat sequence. -Minisatellite binding with locus-specificity and high versatility fragment acceptability Natural consensus with - complete or incomplete tandem repeats derived from the core sequence isolated or artificially created.

The core components of a probe can be defined by various methods that expose the same basic principles. most The basic principle is that the repeat sequence of the probe is a miniaturized version of human or animal genomic DNA. Consisting of nucleotide sequences from a common core region common to the satellites, or This should be included. This common core sequence has one minisatellite and another In the sense of showing a high degree of consensus, for example 80% or more, between It is “common”.

These minisatellites are generated by, for example, the myoglobin gene 33 bp repeats. 1λ33-positive” fragment, hereinafter referred to as “1λ33-positive” fragment. Detection is achieved by obtaining hybridized fragments. these fragments and miog The 33 bp repeat of the Robin gene contains approximately 16 bp of consensus core sequence.

This λ33-positive fragment was used to form other fragments that also had a common core sequence. It can be used as a probe for the genomic DNA of It may also be accompanied by some small change in.

Another principle is that the core nucleotide sequence is a minisatellite region of the Zamburu DNA. be not so short that it cannot effectively hybridize to the For example, the 33bp tandem in the miniglobin gene. This is very similar to Fuku. Generally, the core is a 6 nucleotide or minisample. The maximum found in the common core of telites should be about 16. probe The repeating array need not consist exclusively of cores, but at either end of the core array. May contain a small number of flanking nucleotides.

Repeating units may vary in terms of either the number or type of nucleotides and the number or types of repeating units. There is no need for exact repetition of either the core or non-core components. but However, even though this is an approximate term, it is used in this specification as a repeating unit. It is convenient to describe and define the A block of n repeat units can be It may have any nucleotide sequence at the end (the extent and type are usually irrelevant). I am in charge.

Polynucleotides of this invention are defined in any of the following ways: Especially inclusive.

No.” υ’fJL The following general formula % expression % (1) read in the meaning of 5'-3' [During the ceremony, "Core" refers to a sequence having at least 6 contiguous nucleotides; The nucleotides in the following sequence are read interchangeably: GGAGGTGGGCAGGAXG (2) AGAGGTGGGCAGGTGG (3) GGAGGYGGGCAGGAGG (4) T (C) I, 1GGA GGAXGG (G), C (5A) T (C), GGAGG 8 (A) QGGG C (5B), where X is A or G and Y is C or T, m is 0.1 or 2, p is 0 or l, and q is 0 or 1. , n is 31 or more; J and K together represent 0 to 15 additional nucleotides within the repeat unit; ;and, H and I are each 0 or more additional nucleotides that flank the repeat sequence; : and that: (i) “core” and J and K are each (J, core A, K) repeat units do not necessarily have the same sequence or length; (ii) “Core” can also refer to different core sequences; (iii) The actual total core sequence in all n repeat units is equal to the same number n repeat units. 70% or more of the “true” total core sequence defined above for equations 2-5 in having the same sex; provided that a polynucleotide having: and A polynucleotide having a sequence complementary to this.

Star-shaped small Z bag The following general formula: % formula % (1) [During the ceremony, “Core” means 6 to 16 consecutive nucleotides that have the same meaning of 5’-3’. (1) myoglobin tandem repeats of approximately 33 nt per repeat unit; Searching human or animal genomic DNA using probe DNA containing the sequence 5′=3′ common core of the first human or animal minisatellite obtained by (2) a tandem reaction region comprising a common core region of the first minisatellite; Exploring human or animal DNA using probe DNA containing a duplicate sequence 5'→3' common core of a second human or animal minisatellite obtained by and (3) a common core region of a second minisatellite. Searching human or animal genomic DNA using probe DNA containing repetitive sequences 5' = 3' common of the third human or animal minisatellite obtained by selected from the core region, each tandem repeat sequence described above is a repeat of 3 or more units. ] a polynucleotide having a complementary sequence; Chido.

Tamashiku Sadago The following general formula: % formula % (1) [During the ceremony, “Core” is a human or animal body that exhibits a consensus of 75% or more, preferably 80%. Six or more consecutive sequences from within the common core region of the minisatellites of the genome DNA of an object. represents any sequence having nucleotides; The “core” does not necessarily have the same sequence in each repeat unit, and All symbols in are as defined above] a polynucleotide having a sequence complementary to this; Do.

Eto■Definition The following formula: % formula % (6) (where P is not G, W is A or T, and X is A or G) Consecutive (5'→3') cores selected from the array represented by or its variant - a polypeptide with three or more repeats of a 6-35 nt sequence (all repeats) The actual total core sequence in is defined for equation (6) in the same number of iterations. (provided it has at least 70% homology to the “true” core sequence); and a polynucleotide with a complementary sequence thereto.

In the above formulas, and throughout, sequences are denoted by the conventional notation 5'→3'.

This invention relates to DNA, RNA, and any other material that can hybridize with DNA. It includes polynucleotides of the following types.

The polynucleotide defined above is unlabeled and double-stranded (ds) or single chain (ss) form.

This invention provides labeled polynucleotides in the ss form for use as probes. tide, and their labeled precursors from which to make ss-probes. Includes things that can be done.

These are preferably 33p-radiolabeled in any conventional manner, but As an alternative to this method, other methods (known in hybridization techniques) may be used. can be radioactively labeled by means of he 19811CN-UCLA Symposium on Develo pmental Biology using Purified Genes , Keystone, Colorado, March 15-20, 1981, VolX 1981.647-6 D. Brown et al., eds., or by the method of CyWard et al. or similar species.

D. B. Malcolm et al., Abstracts of the 604th Btochemical Society Meeting, Cambridge, UK It can even be enzyme labeled by the method of the Japanese government (Meeting of July 1, 1983).

According to another aspect of the invention, a sample containing human or animal DNA is Polynucleotide probes are provided that are useful in determining the origin of genes in contains labeled or marker components and limits the sample DNA. fragmentation into corresponding DNA fragments obtained by endonuclease fragmentation. of the human or animal genome to an extent that allows hybridization of the probe. Three or more tandem repeats of sequences homologous to the minisatellite region (including variants) comprising a polynucleotide comprising: : a) Each repeat is the same repeat present in multiple minisatellites from different genomic loci. Consensus of similar length - contains a core that is more than 70% homologous to the core region; b) the core has a length of 6 to 16 nucleotides; C) the core has a length of 6 to 16 nucleotides; The total number of nucleotides within the repeating unit is 15 or less; It is characterized by

The invention further includes a method for identifying a sample of human or animal genomic DNA. , this method searches the DNA with the probe of the present invention, and detecting hybridized fragments.

This aspect of the invention: Fragment total DNA from a sample of cellular material using restriction endonucleases death, Highly variable DNA fragments are added to labeled or marker components into the repeat core. hybridize with a probe as defined above containing a component, and Binds to DNA fragments of different lengths or more generally to bands of different molecular sizes Typically, the fragmented DNA is Before ization, fractionation or separation according to chain length, e.g. by electrophoresis, is performed. to detect marker concentrations whose individual components are characteristically specific to genetic origin. Get the pattern.

definition The following definitions used throughout this invention and the foregoing description may be of assistance.

The trend is negative The length of a region of human or animal DNA at one recognized locus or site or a sequence is said to be hypervariable if it exists in many different forms. It will be done.

Ij One evening punishment (RFLP) This refers to the human or animal matter that is separated after electrophoresis and detected by a probe. Genetic diversity in the patterns of DNA fragments.

mini satellite It consists of human or animal DNA consisting of tandem repeats of short DNA sequences. It is an area. It is not necessary that all repeat units exhibit exact identity.

(The probe of this invention comprises a mini-satellite that is atomic in nature.) Genes that differ from individual to individual or other segments of DNA are heterogeneous It is said.

Yueni” μ Katana Riichi Originally used to mean consensus-core sequence, but derived from Extends to any repeated or modified array.

And/Marusuka each-new 7-<μA month There is no substantial difference between repeating units of two or more minisatellites of different origins or genetic loci. is a sequence that can be identified as an exact match.

Repetition 1 - Complete or incomplete tags for a given core sequence or segment containing a core sequence It is an array that is a double repeat.

undefined core (array Y- A core sequence that perfectly matches one of 2 (2) to (8) within its own length .

Hengetsu Funeral” Tanni μ sword 91 An actual core that is slightly separated from the defined core sequence (more than 50% homology) It is an array.

A sword and a sword Accurate tandem replication of a given core sequence or of a segment containing a core sequence It is a barrel array.

・6 all' (arrangement) at least one unit has at least one other base pair substitution and/or length This is a medium to different arrangement. (usually at least 3 in one probe sequence) tandem array of at least one defined core within the probe sequence. There will be an array and at least one variant. )■Kake X When comparing two sequences of the same length, the difference in the number of base pairs (bp) and the number of bp substitutions This is the percentage of the subtracted number of bp.

The term 1~human±I'Cnt) and base pair (bp) are used interchangeably. Both are DN It can be applied to A or RNA. Abbreviations C, A.

G and T are (deoxy)cytidine, (deoxy)adenosine, (deoxy) as usual. ) Guanosine, and either deoxydemidine or uridine.

A tandem repeat sequence (artificial or natural, Qj isolated) can be a complete repeat. However, more preferably it is an incomplete repeat. Preferably at least 2 The repeats are incomplete repeats of the consensus-core sequence. Preferably 3 or more repeats, and more preferably seven or more repeats, are present in the probe sequence.

Probe production involves isolation of natural minisatellites by cloning and DNA sequencing. Identification by column determination can be included.

This also leads to the required core cutting and its subsequent tandem iteration. or the facilitation of non-identical exchange with fragments of different origin. child This can also include cloning of polynucleotides.

On the other hand, the construction step involves the synthesis of the identified consensus-core sequence or fragments thereof. You can The consensus-core sequence preferably contains 6 bp or more. It preferably contains 16 bρ or less. Next, create tandem repeats of the synthetic core do.

Needless to say, the polynucleotide may be a successful or partially successful intermediate. Cloning can be the result of a sequence of operations at different times after cloning, and so that the final polypeptide has slight resemblance to the parent minisatellite. Fragments of original or synthetic origin can be included.

The probe of this invention is useful in the following fields.

1. Paternal and maternal testing in humans.

2. Establishment of family groups, for example in settlement disputes and inheritance disputes.

3. Zygasity test in twins.

4. Tests on consanguineous marriage in humans.

5. General pedigree analysis in humans.

6. Identification of genetic disease loci in humans. This detects genetic defects It becomes possible to create highly specific probes.

7. Forensic medicine (a) Identification of semen from rape victims, (b) E.g. soiled clothing identification of blood, hair and semen from (c) Identification of human remains.

8. Research on cell chimerism, e.g. bone Tracking donor versus recipient cells after spinal cord transplantation.

9. Livestock breeding and pedigree analysis/certification. (This applies, for example, to the routine regulation of purebred animals) and inspections, as well as pedigree inspections, for example in litigation cases related to race horse and dog breeding. could be included. ) Furthermore, it shows the relationship with economically important genetic traits. To provide a genetic marker for deafness.

10. Checking for contamination of pure cell lines and E1 extenuating work, cultured movement Daily quality control of product cell lines.

11. Analysis of tumor cells and tumor molecular abnormalities.

12. Polynucleotides or probes derived from them have potential in plant breeding. It is expected that it will have many uses.

Shell page tyv1 ■V mouth W skin Figure 1 shows that the 33hp repeat sequence of the myoglobin gene is inserted into a plasmid and This is a schematic explanation of the manufacturing method for cloning it.

Figure 2 shows fragments of genomic DNA that hybridize to various DN A blows. It is a photocopy of the autoradiograph photograph of The pedigrees of related individuals identified in the two autoradiograms are shown.

Figure 3 shows unrelated individuals probed by three different probes of this invention. Autoradiographs of DNA samples from three human individuals are shown.

Figure 4 shows DNA from nine large human individuals probed with the probe of this invention. Showing autoradiographs of A samples, some of whom are related, and 2 Humans are identical twins.

Figure 5 shows two human and 17 non-human subjects probed with the probe of this invention. Showing an autoradiograph of a DNA sample from an outside animal. Figure 6 shows a Ghanaian family caught up in a settlement conflict that was explored according to this invention. Figure 2 shows a series of autoradiographs of DNA samples from members of the group.

Figure 7 shows an autoradiogram of a DNA sample from a relative affected by neurofibromatosis. Show rough.

Figure 8 shows possible co-transfer of minisatellite fragments and fetal hemoglobin. A Gujarati bloodline created to test for the genetic presence of human beings (HPFI+). The autoradiograph of their DNA is shown.

Figures 9A and B show HPFH and various minisatellites in large kinship relationships. This is a genetic diagram showing the inheritance of .

Figure 10 is a diagram showing the preparation of cloned artificial minisatellites. be.

Figure 11 shows the embryos of two unrelated individuals probed with the new synthetic probe. A series of autoradiographs of DNA samples from the disc is shown.

Figure 12 shows a series of autoradiations of searched DNA samples from large kinship relationships. Showing Ograph.

Figure 13 shows the various D N obtained for twins using two different probes. 1 is an autoradiograph showing an A-band pattern.

Figure 14 shows band patterns formed using single-stranded probes and double-stranded probes. This is an autoradiograph to compare.

Figures 15 and 15A are audio charts showing DNA fingerprints obtained from forensic samples. It is a traradiograph.

Figure 16 is an autoradiograph showing DNA fingerprints obtained from a family of dogs. .

Figure 17 is an autoradiograph showing DNA fingerprints from a family of short-haired domestic cats. be.

Figure 18 shows DNA fingerprints obtained from various sheep using two different probes. This is an autoradiograph shown.

Figure 19 is an autoradiograph showing DNA fingerprints from three different pigs. Ru.

Figure 20 shows an autoradio showing DNA fingerprints for a family of cows and additional cows. and Figure 21 is a graph similar to Figure 20 using different probes. It is a traradiograph.

Kouni% (sha) awei 10-20kb of human DNA cloned into phage λL47.1 A human genomic library of the au3A portion was used as a 33bp myoglobin repeat probe. "Screened by hybridization with pAV33.7. 3X 40 or more strongly or weakly hybridize in a library of 10' recombinants A plaque was identified. A random selection of 8 of these positive plaques was purified. λ33.1-15), and using Southern blot analysis of phage DNA, The DNA that hybridizes in each recombinant is the unine short (0,2 to 2) of the recombinant. It was shown that it is located within the kh) region. Sequence analysis revealed that each of the eight recombinants This region contains minisatellites consisting of 3 to 29 tandem copies of repetitive sequences. The repeat sequence length ranges from 16 bp in λ33.15 to 64 bp in λ33.4. It was shown that the Most minisatellites contain an integral number of repeats. Ta. Next, at λ33.6, the 37bp repeat is divided into basic 12bp units. It consisted of a branched trimmer. Each λ33 recombinant has a cleft between each minisatellite. representative of different regions of the human genome as determined by loan-specific DNA sequences. Ta.

Eight cloned minisatellite regions were isolated in a Hinfl digest of human DNA. 0.5 smaller than the β-type 2-6 kb DNA fragment that can be detected by AV33.7 It was located within the ~2.2 kb Hinf I DNA fragment. Cloned Mi To determine whether any of the Nisatellite regions are abundant, Bellylated single-stranded DNA probes were extracted from appropriate M13 subclones of each minisatellite. Fourteen unrelated British genes were prepared and digested with HtnfI with high rigor. The DNA was hybridized to a panel of Caucasian DNA. Typical hybridization The hybridization pattern indicates that each probe is human under these hybridization conditions. Detection of unique regions of the genome and three of these regions are highly abundant .

Further details of the above procedure are provided in the examples.

Now, with respect to the above definition of polynucleotide, the definition has the following general formula: % formula % (8) (In the formula, C indicates the core sequence, and the other symbols are as above.) matches. In all definitions, the core array of one unit is the same as But it can be different. For example, a consensus that applies to a repeating unit as a whole Contains 1 or 2 missing nucleotides compared to the sequence, and contains 1 or 2 nucleotides even if it lacks a nucleotide or differs by (for example) 1 to 4 nucleotides good.

The core can be defined in a variety of ways. The general definition is that the core sequence can be identified This can be obtained by referring to the steps that can be taken. genomic DNA satellite For example, compare the region with a sequence such as GGAGGTGGGCAGGAXG (2) be done. selected from all possible X-nucleotide length sequences contained in formula (2). The X-nucleotide taken from the minisatellite that shows the greatest homology to the X-nucleotide The cleotide length sequence is considered to be the core sequence. Needless to say, this is the core This is a very narrow way of defining length, one or a few seven nucleotides in length, and up to 16 nucleotides in length. (There are likely to be a few). For a core defined like this These cores are diverse to the extent that all n repeat units have an average homology of more than 70%. It is assumed that gender can exist. Flanking sequences J and K within the repeat sequence are They are not included in homology calculations and are only compared between cores. Defined code Ars can have different lengths and can be "mixed." In other words, how many In some repeating units, for example, it is homologous to GGGCAGGAXG of formula (2) In other respects, it is homologous to, for example, GGGCAGGTGG of formula (3). follow Therefore, the deformed body is not necessarily homologous to the “core” itself, but is homologous to (core) n. It should be considered as homology.

The “first” definition defined earlier derives from the above considerations, but is not indicated. (2) to (5) Width, depending on the circumstances, any length from 6 to maximum 12 to 16. It is simplified in that the core is defined as an array. Similar for deformed body Regulations exist.

The second definition consists of consecutive high Define core as the hybridization stage. A wide range of human genomic DNA Perform a sufficient number of hybridizations on the library to obtain a sufficient number of hybridizations. The hybridized fragments are tested, probes are made from these, and the genomic DNA is recombined. By searching 7 and theoretically doing this an infinite number of times, the mini-satellite DN It is possible to arrive at a range of consensus core sequences widely represented in A. It will be easily understood that In fact, in a sufficiently wide consensus core area These operations must be performed very many times and on a large scale to reach It was not expected that there would be none, and therefore (arbitrarily) only three search operations were included in the definition. included.

In the “third” definition, W represents the core and 25% (e.g. 16 4 nucleotides in the nucleotide), a widely shared copolymer with no different diversity. Relying on the possibility of sensor score area. Approximately with a possible variability of less than 25% If the core region of 16 or more nucleotides is defined in this way, the core W can be defined as It is defined as a sequence of 6 or more contiguous nucleotides from within a region.

“The definition of the 4th degree has been redefined from research on synthetic polynucleotides. Contains a set of consensus cores (6).

These studies lead to other definitions of shorter core sequences.

Therefore, preferably the contiguous (5'-3') sequence: PGGGC<G(7) is conserved in all repeating units, where P and W are given in the fourth definition above. It has an acquired meaning. P is preferably T and W is preferably A.

Preferably also a continuous (5'3') sequence: TGGGCA (8) is conserved in all repeat units.

According to another point of view, consecutive 5'-3' core sequences: GGPGGGCWGGWXG (7) (wherein P is not c-cit, W is A or T, and X is A or G) be) polynucleotides having three or more repeats including The “true” sum defined with respect to equation (7) during the same number of iterations (provided that it has 70% or more homology with the core sequence), and the above A polynucleotide having a sequence complementary to is provided.

In all the above definitions, the core is at least 6 nucleotides long and More preferably 7 or 8 nucleotides, and most preferably 12 or more. For example, it is 14 to 16. Arrangement of 2 (2) GGGCAGGAXG ( The terminal 10 nucleotides at the 3' end) have high consensus and are particularly promising. It seems so.

The deformed core is preferably 70% or more, and more preferably 80 or 85% % or more homology.

Flanking sequences J and K within each repeat unit are preferably omitted at each end; or shorter, e.g. 0.1 or 2 nucleotides, and preferably J and K together should not exceed 20 (and more preferably should not exceed 20) It's not possible. The total number of nucleotides in the total of J + core ten within the repeat unit is preferably preferably greater than 36, and more preferably greater than 31, and most preferably greater than 25. Shouldn't.

The number n of repeating units is preferably at least 1o, conveniently from 10 to 4o, but However, in principle it can be any number, even up to 10,000. be.

Flanking sequences 11 and I5 are unrelated. these can be omitted , or present as any number of nucleotides, e.g. up to 20,000 However, it is not very wise to use such long probes. repetition Even if the sequences are 5s-DNA, they may contain cps-DNA. can.

The identification method can use any known search technique, the most common of which is The ability of sample DNA to not cleave tandem sequences or their ability to be explored Restriction enzymes (if appropriate, use 1 cleavage according to the type or types).

Anal Hitomio Sotoda Okinin' City J Shihoden transfer l Shirimi G self mu tile restoration father Ushitsukura 1 to Tai 1 hole Nizu Near Court.

A schematic diagram is shown in Fig. 1 showing the five steps in the construction of this probe shown in (a) to (e). It is explained inside. The starting myoglobin gene (a) is P, Weller et al., E ? IBOJ, 3.439-446 (1984). shown there As shown, the gene is sandwiched between almost identical 9 bp sequences (r in Figure 1). A region located in the first intron consisting of four repeats of a 33 bp sequence have This region was isolated into a 169 bp fragment (b), which was subjected to end modifications. by reproducing and cloning into the Sma1 site of plasmid pUCl3. (J, Vieira et al., Gene 19°259-268 (1982 )checking〕. The third and third restriction endonucleases (A) Limonomer was isolated by cleavage of the fourth repeat (c). (Iterations 1 and 2 One-group substitution in removes this site and forms a DdeI (D) site in its place do. ) non-identical vaI [anti-identical by ligation of 33 bp monomers via cohesive ends; A head:tail polymer (d) of unknown number of functional units was formed. Contains at least 10 replicates The polymer containing p is isolated by preparative agarose gel electrophoresis, the ends repaired, ligated into the Sma1 site of UCl3 and cloned into E. coli JM83. (See J. Vieiva et al., supra). p AV33.7 occurred. The structure of the polymer DNA insert in plasmid (e) was constructed using amHI + Eco Cut out the insertion part in the polylinker using R machining, and α-3! to P-dCTP Fill-in labeling at the amH1 site and human vaIr Confirmed by partial digestion.

Labeled partial digestion products were separated by electrophoresis on a 2% agarose gel. Ta. p AV33.7 is 767bpBamHI once coRI as shown in (e) It was found to contain 23 repeats of the 33 bp monomer contained within the fragment.

(2) Minisatellite regulation of human tomato by myoglobin 33b probe Placement of ゛　 10-20kb human DNA cloned into batatteriophage λL47.1 A library of A fragments (P, Weller et al., supra; and W, A, M, Lennen et al. , Gene 20.249-259 (1980)), P.

32p-labeled by the method of Weller et al., supra, in step (1) above. By hybridization with the 767 bp pAV33.7 insert described in was screened. A selection of 8 positive plaques was purified to λ33.1-1 A recombinant designated as 5 was obtained. Each of these phage DNAs was combined with 1nfI or digested with AEM, electrophoresed through a 1.5% agarose gel, and The 33-repeat-related sequences in The location was determined by hybridization. Each recombinant has one “λ33− Positive"1 button fl and 2 fragments were given, but λ33.4 and 11 were detectable units. did not give aeI[I fragment (Haell in the repeat region in these recombinants) (due to the presence of cleavage sites). Separate λ33-positive Hinfl and aeIn fragments isolated by gel electrophoresis, optionally repaired, and duplicated by DNA A blunt end was ligated to the ma1 site of M133mp8 (J.

Messing et al., Gene 19.269-276 (1982)), E. The dorsal corner 55-M13 recombinant transformed into E. coli JMIOI was separated and arranged by dideoxynucleotide chain termination method. The row has been decided. All λ33-positive fragments contain tandem repeat regions; This could be directly sequenced in some cases. Repeated regions are sequenced In other cases too far from the imma site, insert the M13 insert with restriction endonuclease. was shortened by cleavage and resequenced.

The structures of the λ33-positive fragments are λ33.1, 33.3, 33.4, and 33.5.

Later in the form of eight maps designated 33.6.33.10.33.11 and 33.15. Shown below. The actual restriction enzyme and nucleotide fragment length used was λ33. 1: Danki ■, 2000; 33.0: 1 button fl, 465; 33.4: and inf I, 2000; 33.5: Dan in f I, 1600; 33.6 :1 wear■, 720; 33.10: Danki■, 720; 33.11:1 button f I , 1020゜ccag ta tcc tgggaa t tcg taa tca tgg tca tagc tg t tcc tg tg ■≠■ Lambda 33.10 a gacac tgccc tgg tgBc tc tgacceec t gcagcc tce ta tge tc ta---tgc t tg gggca gcac tcacacacag taag tgcceaag tcaaa tgaaa tacctacaga tgagg tg tg t gc tta t tc tc tg tgcaaga t t tcagag @t (3) The ox [core The repetitive sequences of each region of ``West 1'' and 6 were analyzed using dot matrix analysis. Myoglobin 33bp repeat sequence in AV33.7 and its reverse complement compared. Very prominently, the myoglobin 33 bp repeats and the λ33-positive fragment One small clear region of sequence similarity was found between the consensus repeat sequences and Ta. The same region is shared by all eight λ33 fragment repeats, and It will be called the “common core.” The chart below shows the comparison.

In this chart, each consensus sequence determined in step (2) above is shown in its entirety. These include myoglobin 33bp probe and human This is a tandemly repeated portion in a λ33-positive fragment isolated from mu DNA.

This chart shows the common core region as 16 nucleotides long in uppercase script. show.

Common core area *This sequence is a trimer, so the flanking regions contribute to the core.

Again, X is A or G, Y is C or T, and - is the missing nucleotide. be. There is a substantial degree of identity for these 16 nucleotides, of which 8 indicates a 100% match, and the 9th “X” is either A or G. Match within. These nine nucleotides are underlined in the bottom row. , indicating the nucleotides of the common core region.

The tandem repeat residues indicated in lowercase script are present at the edges of the common core region. Exists. The beginning/end of each iteration consensus is identified by the symbol ma; λ33.4 and and λ33.15 there are non-integer repeats and therefore separate The start and end of the loop are indicated by different symbols. Common core areas are subject to complex analysis. It will be recognized that only one could be identified.

Because this often results in a single consensus sequence of λ33-positive fragments, the entire does not belong to the myoglobin gene, and two contiguous sequences of the myoglobin gene 33bP repeat. This is because they are eager to do so.

Degree of identity with polynucleotides defined as falling within the scope of this invention To explain, various determining factors are shown in Table 1 below.

(4) Polymorphism ``Yotonigeno expression Nmu pulse piece j individual p section 332'' taste characteristic 4 and bu'' Sn-: Boo ζ-, A ini 4. Gino ζ-I-2-2-Kushin' Goyo- Regarding the 2nd figure of the 4s-y outbreak, British Nikasas A random sample of people (1-6) and a large Anglo-Asian family (7-18) were selected. DNA was prepared from leukocytes collected from the members. For convenience, pedigree indicates male. It is indicated by a square, a circle representing a woman, and a marriage by a line between them. cousin The two close marriages between the parents are shown by the two tree lines between the parents. A 10ug sample of DNA was Digested with 4nfI and electrophoresed through a 20<em>long 2% agarose gel. Sartorius denatured in situ and plotted Transferred to a nitrocellulose filter.

A single-stranded 32p-labeled hybridization probe was transduced into a minisatellite ( was prepared from an M13 recombinant containing the Kundem repeat region. EXACT PROS USED The details will be described later.

The procedure was as follows. Approximately 0.4 μg of single-stranded DNA was sequenced with 4 ng of Tsukumar. Fixed Primer Group, Dock Orth, etc., Nucleie Acids Res , 9, 1691-1706 (1981)) and 10ttl of 10mM 1'1g cff2,’10+nM Tris-14Cj! (pH 8,0) at 60℃ It was annealed for 30 minutes. Primer extension was performed using 16μ2 of 80uM dATP. , 80, ljM dGTP, 80 μM dTTP, 10mM Tri s-11C! ! (p)[8,0), 0.1mM EDTA and 3pE (30u C3)ir-”P-dCTP(3000Ci mmole-’) and 1. crj? 5 units μl of -’Kleno-fragment (Behring) was added and incubated for 15 minutes at 37°C. Nobu The length is 2.5ttl! of 0.5 mM dCTP and an additional 1 at 37°C. Completed by up-linking (chasing) for 5 minutes.

"Chasing" is on the mold of M13ss DNA This means adding a dNTP mixture to complete the dsDNA ring.

The DNA is placed in a suitable M13 polylinter in the insert or distal to the insert. Limit End Nuku! /A - Cleaved at the upper part, 1.5 of 1/10 volume M Na11 (, denatured by adding 0.1 M DKTA and plate A 1tlr piece of 32p-labeled single-stranded DNA extending from the imer was added to a 1.5% low melting point aliquot. The cells were collected by electrophoresis using a galose gel (Sea Plaque). cut out The band (specific activity>109CpH1/,!jg DNA) was deprived of 1mg of alkali. Share carrier human fetus Qi D N A (0,3 MNa0 14, 20 mM EDTA at 100°C for 5 min, then resuspended in H (J). The mixture was melted at 100°C in the presence of added directly to the sample. Carrier DNA can also be used for subsequent hybridization to repetitive DNA sequences. It also helped to suppress hybridization.

The exact hybridization probe used was (A) 33.1, minisatellite. Light (26 repeats of 62nt sequence - 1612nt) + approx. 360nt of furan Approximately 2000 nt subcloned Haell fragment containing proboscis human DNA; ( B) 33.4, 2015nt) Minisatellite contained in the fin f I fragment 695n on the proximal side of the primer of the non-minisatellite EcoRI fragment; and (C, D, E) 33.15, λ33.15 minisatellite array (shown above) (29 repeats of a 16 nt sequence, differing by an average of 2 nucleotides from the common core region) 592nt subcloned fragment containing +128nt flanking human DNA, Met. A, J. Jeffreys et al., Ce1121, 555-564 (1 Hybridization was performed as described by (980).

However, in order to reduce bank ground labeling, dextran sulfate was added. % (W/V) polyethylene gelcol 6000. Filter A and B were hybridized overnight at 65° in 0.5 x ssc, and 6 Washed in 0.2X SSC at 5°C. Filter 〇-E is hybridized and washed in Ixssc at 65°C. Tangs with fixed filter Autoradiographed for 1-3 days at -80°C using a thenic acid enhanced screen. did.

As shown in Figure 2, repeat core probe 33.15 was digested with and inf Detecting the extremely complex profile of hybridizing fragments in human DNA Ta. Only the largest (4-20knt) auxiliary fI fragments can be well resolved, and these The hybridization profile provides a solid-specific DNA “fingerprint” He showed extreme versatility.

Pedigree analysis shows that even among single blood relations (16-18 in Figure 2) of cousin marriages, Extremely variegated diversity was confirmed, to the extent that all individuals could be identified. In Figure 2 Families < D, E) have most of the large Hinfl fragments from each parent to some of the offspring. This suggests that most of these fragments are only transmitted by heterozygotes. The heterozygosity of these large hypervariable fragments is 100 % must be reached. In contrast, all fragments in the descendants A fragment can be attributed to one or the other parent (with one exception), and It therefore provides a stably inherited genetic marker for 1 cent. specifically from my father Bando Somo Inu passed on to sons or from mother to daughter (filter D, (See Figure 2). This excludes the Y and X linkages, respectively, and this This means that these minisatellite fragments are primarily derived from autosomes. this It is not yet known where these DNA fragments originate from in the set of autosomes. However, these do not originate from a single region of one autosome. Not so , pairs of parental fragments that segregate independently in the progeny can be identified (filter D, Fig. 2). checking). To be exact, if there are at least 61 AB or -1 descendants, then 1 The pair of bands AB in one parent (and missing from the other) are allelic cannot exist; the existence of A- or -3 recombinant progeny further suggests that there is a close link between A and 8. Establish lack of linkage. The autoradio log of the family shown in the second diagram. Rough's Attention Search) Consideration of at least 10 in the mother by those criteria. exhibiting separable bands, eight of which are mutually non-allelic, and Not closely linked. The other two bands each contain one of the eight unlinked fragments. progeny, where only the 8- and -B progeny are analyzed. observed only in a limited number of grandchildren, although such a small sample is not sufficient to prove that a pair of fragments such as are alleles at a single locus. stomach.

In conclusion, the core probes can provide useful information at the same time. This conclusion is further detailed in Example 8. will be considered.

On the other hand, the other two probes (filters A and B, FIG. 2) are according to the invention. However, it gives only one or two bands, and many different β-type regions are the same. sometimes cannot be detected and therefore cannot be used for general diagnosis .

Banmata A probe for determining the state of human DNA. ■ Cloning of core λ33-positive fragments λ33.5 and λ33.6 Two other probes derived from the following were prepared similarly to Example 1, step (4).

33.5 probe is a 308 nt DN A cloned into M13mp8 fragment and comprises 14 repeats of the above consensus sequence, which consists of 7 It is a 17 nt long variant of the common core sequence with 0 nt flanking human DNA. Ru. 33.610-b contains a 37 nt sequence of 18 repeats, this sequence is now common 3 repeats of approximately 12 nt truncated sequence from the core region + addition at its 5'-end TC. 18 x 17 nt repeat block in 97 nt human DNA I was caught between them. The structure of the 37nt sequence can be expressed as follows.

TGG AGG AGG GGC TGG AGG A-G GGC (or TGG AGG AGG G-C) TC CGG AGG AGG GG The probe was similarly cloned into M13mp8.

In the later description, the 1int consensus sequence AGGGCTGGAGG is used for this protein. Given for robes.

Both probes were labeled with 32p and 14 as in Example 11 step (4). were hybridized to human DNA from a panel of unrelated Caucasians. both The probe detects a complex set of hybridizing fragments, many of which are extremely showed a β-type change. Some fragments detected by 33.5 are novel. , and has not been previously detected by the 33.15 core probe. Ru. 33.6 probe detects almost entirely new minisatellite of St. , and their correct inheritance was proven through pedigree analysis. (See Example 8) thing).

The following examples further explain and illustrate the invention.

Digestion of the sample DNA is preferably performed using a restriction enzyme that recognizes four base pairs of nucleotides. This is done using endonuclease.

4bp recognition limit where DNA fingerprint pattern for longest hypervariable fragment is used It was found to be thick (independent) from the endonuclease. These large fragments are not derived from longer minisatellites, and each but lacks restriction endonuclease cleavage sites and has a normal dense 4 bp cleavage site. Containing full-length uniform minisatellites sandwiched between DNA strongly suggests. This means that most of these large minisatellite fragments are linked In the above examples and examples showing that they are not divided into lineages and independently divided into lineages. This is consistent with the results shown in .

In a preferred embodiment, the DNA sample is separated into two separate samples resulting in two different fingerprints. In the hybridization of two different probes, e.g. lambda33. Double fingerprinting is performed using probes from the 15 and 33.6 fragments. two unrelated people The already low probability that related individuals have the same fingerprints is further reduced by this measure. Ru. For example, Example 4 provides an example of an incompletely dissociated hybrid, preferably 4 kb terminal in length. It shows a probability as low as 10-19 even when soybean DNA fragments are ignored.

The invention includes a method of paternity testing. Approximately half of the β-type minisatellite fragments in offspring These paternal fragments are derived from the father, and these paternal fragments are determined by comparing the DNA fingerprints of the mother and child. can be identified. All of these paternal fragments are normally present in the father's DNA. There will be. Using probe 33.15 and a DNA fragment with a length of 4 kb or more, A hypothetical father has all six paternal-specific DNA fragments typically identified in his offspring. The probability of having by chance is of the order of 10-' and both probes 33.6 and The use of 33.15 is expected to reduce it to the order of 10-s. .

Of course, the exact probability depends on the exact separation and the complexity of the DNA pattern obtained; Additional paternal fragments less than 4 kb in length are then analyzed, or a third probe is used. If possible, it will be improved.

Because of the DNA fingerprint, one drop of human blood is sufficient to obtain NA (0.5 to 5 micrograms). can be rapidly isolated.

Then, the DNA of the individual whose fingerprints have already been collected includes 2 people + 2 sisters. DNA fingerprints were created from a random panel. two pre-characterized individuals The body can be easily and unambiguously identified based on DNA fingerprint comparison, and a substantial number of The same was true for two sisters who share a minisatellite fragment of .

DNA can be taken from different cells of a given individual, all giving the same fingerprint. I can do it. Therefore, no DNA fingerprints can be identified for sperm and blood DNA, and - ovoid twins. The pattern for children is similar. In addition, the DNA fingerprint of blood DNA and the origin of the same individual Limboblastoid cells transformed with Epstein-Barr virus The pattern is similar to that of cultured cells, as shown by comparison with DNA isolated from lines. It also appears to be maintained in cells.

Non-human animals to which this invention can be applied include most mammals, birds, and other animals. Includes tanks and fish. For example, chickens, hamsters, rabbits, mice, sparrows, They are snails, frogs, newts, and fish. In the case of chickens, hybridizing DNA Very complex blurred parts of the fragments were produced. Digestion by Haem is “clear” The blurred parts were removed leaving the fingerprints. Therefore, chicken DNA also contains its repeating units contains a long core-containing satellite containing one or more a<em>cleavage sites. , therefore cleavage by HaelI [removed to the bottom of the gel during DNA electrophoresis] It is thought that this satellite is reduced to very small DNA fragments that are Tokio other animals produce indistinct bands, and these cleave longer fragments. 4) If the NA is digested by a suitable enzyme, it will become separable. it is conceivable that.

In the following additional examples, the temperature is in °C.

Example – turtle By A, J., Jaffreys, Ce1118-11-L　O (1979) DNA was isolated from fresh human placenta as described. three individual Placenta was used and labeled 1-3. I) A sample of 8 microkum dalam of NA, Hinfl and and/or Σ憇3A, and recovered by ethanol precipitation after phenol extraction. Then, it was passed through a 20 cm long 0.6% agarose gel at 30 V for about 24 hours. ．． Electrophoresis until all DNA fragments less than 5 kb in length are removed from the gel. did. Next, the DNA was plotted on a sartorum-nitrocellulose film. Moved to filter. High pressure activity (109epm”P/microgram DNA) ) Single-stranded M13 DNA, probe as described in Example 1, Step 1. Prepared. The exact probe used was = (a) λ33.5 minisatellite ( 17 nt x 14 repeats) containing approximately 60 nt flanking human DNA. 220ntHa, subcloned into the Sma■ site of M13mp8, consisting of 33.5 probe consisting of eIII D N A fragment; (b) minisatellite + A subcluster at the Σ=X site of M18mB, consisting of approximately 50 nt flanking human DNA. Loaned, 33.6 probe consisting of 720n and aell fragments; and and (c) the same 33.15 probe as in Example 1, step (4) (this is a minisatellite +128nt flanking human DNA, PstI + Sm5-1 erased 599ntP subcloned into M13mp19DNA stI-AhalII fragment).

Southern blot hybridization and washing at 65°C in Ixssc. , Example 1, in step (4) the filter (>E!7 as described above) I went there. Filters were left open for 4 days at room temperature without reinforcement screens. Traradiographed.

Each probe produces a different fragment pattern, the complexity of which is used It is largely independent of leotide restriction endonuclease. Figure 3 shows the obtained parameters. Separation of polymorphic fragments less than dkb length, showing the turn Hi+BfI+~S'dan 3A improved in double digestion, which accumulates within one or more repeating units. A relatively separate and invariant 1nfl minisate with a U3A cleavage site. Removal of bank ground hybridization possibly caused by light fragments. Based on leaving. In double digestion, the separability detected by probe 33.15 is The number of versatile fragments that can About 20% of the long-digested minisatellite fragments, which probably contain At sacrifice, it can increase from about 15 to about 23 per individual.

u- Human blood collected from a random sample of 20 unrelated white British people. A sample of 8 micrograms of DNA was digested using button f1, and the minizate described in Example 3 was used. Hybrid by Sachin method using Light Probe 33.6 or 33.15 Formed. Each DNA fingerprint (individual A) is compared to the pattern (individual A) in the adjacent gel trunk. The number of bands in A that are clearly absent in B and the number of bands in B) Number of bands with co-migration counterparts of approximately the same autoradiographic intensity was recorded. All results shown in the table below are averages for pairwise comparisons. . Additional weakly hybridized fragments of the evening scene (approximately 6%) in A are present in B. The strong hybridization fragments were found to be highly hybridized. In such a case, the band at A Since it is not possible to determine whether the code also exists in B, The simultaneous bands in 6A and B that were observed were always the same at the LQ minisatellite locus. In the case of an allele, the average probability that the allele in A is also present in B =2(1-qZ, so q=1-(1-x)"2 gives the average allele frequency degree (homozygosity) related to q. In fact, the co-migrating bands in A and B (not knowledge) proportion is derived by chance from the heteroeminisatellite locus, and therefore the average allele Estimates of offspring frequency and homozygosity are maximal and electrophoretic fractions of minisatellite fragments. Depends on distance.

The probabilities shown in Table 1 are for the individual size fragments of DNA indicated.

The most readable part of a fingerprint, i.e. all DNA over 4kb in size. The three numbers must be combined to obtain an overall probability for for example, All fragments detected by probe 33.15 in individual A are also present in B. The average probability is 0. OB”・’Xo, 20’-’Xo, 276-’=3X This is number 10. By two probes 33.15 and 33.6 in A The probability that all detected fragments are also present in B is 5×10−”.

Table 2 33.610-202.8-':1. OO,110,066-105,1±1. 3 0.18 0.094-6 5.9±1-6 0.28 0.1533.1 510-202.9::i,o O,080,046-105,1±1°1 0.20 0.114-6 6.7±1.2 0.27 0.15 type This example illustrates the somatic stability of DNA fingerprints and their use in paternity testing. Ru.

Lymphoblastoid cell line transformed by EB virus and stored in liquid nitrogen The strain was reestablished by liquid culture two years later.

These cultured lymphocytes were washed twice with saline, and the lymphocyte pellet and white DNA from blood cells was analyzed by A, J., Jcffreys, Ce11.18, vol. 1-1. 0 (1979). Sperm collected from semen is SD except that it was treated with IM2-mercaptoethanol for 5 min at room temperature before dissolving with S. produced sperm DNA in the same manner. Digested with HlnfI and probe 33.6 ( 5 micrograms of DNA hybridized in a) or 33.15 (b) DNA fingerprints were prepared as described in Example 3 using samples of .

i juice 1 This example shows how to detect highly polymorphic regions in the DNA of various vertebrates. 2 illustrates the use of the probe of the invention.

Blood collected from chickens, sparrows and butterflies, liver from rabbits and mice, human DNA samples were prepared from carcasses from which the placentas of frogs and the digestive tracts of frogs and fish were removed. 8 μg of DNA samples were digested with Hinfl, whereas chicken DNA was digested with Hinfl. I digested it. Restriction digests were electrophoresed through a 0.6% agarose gel and denatured. , transferred to a Sartorius nitrocellulose filter. , hybridized as described above using human minisatellite probe 33.15. formed a

The stringency of hybridization was 65°C in 1xssc.

The results are shown in Figure 5 for the following samples.

1.2: Unrelated human placental DNA 3: Rabbit DNA from F1 hybrids of Alaska and Vienna White breeds 4: Rabbit DNA from Alaska species 5.6: Mice from two wild-caught Greek mice (Mus mu sculus) DNA7: Mouse from inbred strain DBA-2 (Mus mus) culus) NA 8.1 Mouse from inbred C57/BLIO (Rnusculus) D N A 9.10: Chicken DNA: Note: DNA was digested in 1 hour.

11.12: Sparrow DNA 13.14.15. :Chogenbau DNA16.1’7:Frog (Xenopus) tropicalis) DN A18.19: Small fish DNA A variety of valid DNA fingerprints were obtained from almost all vertebrates tested, and It can be seen that this method provides information to the same extent as human DNA fingerprints.

In addition, chicken DNA'A cleaved with Hinfl is hybridized to NA. Although not very strong, very complex obscurities (not shown) were produced. But long When digested with HaeII, this stain is removed and a clean polymorphic fingerprint pattern is obtained. This caused a problem.

The two inbred strains of mice have simpler fingerprints than wild mice. this is wild Although most hypervariable minisatellite loci are heterozygous, inbreeding , are homozygous, and in inbred lines hybridization reduces the number of NA fragments by half. It is thought that

The following examples further illustrate the application of the particular probes described above.

Example j- In this example, traditional genetic methods are extremely difficult, if not impossible, to solve. The use of DNA fingerprint analysis in difficult immigration cases is described.

This case was born in the UK, immigrated to Ghana to be reunited with his father, and then his mother , a Ghanaian man who returned to the UK alone to be reunited with his brother and two sisters. Concerning boys. However, there are cases where unrelated boys or all of them are living in Ghana. There is evidence to suggest that a swap occurred with the son of one of the mother's sisters. It was. As a result, the boy who returned home was not allowed to reside in the UK. the house At the request of the family's attorney, an analysis was conducted to determine the boy's mother. make things complicated Therefore, neither the father nor the mother's sisters were used in the analysis. Furthermore, the mother said that the boy She was sure it was her son, but she was unsure about his father. won. Therefore, available family members (mother M, brother B, sisters S1 and Sl and and DNA fingerprints from blood DNA samples taken from the boy in question , detecting different combinations of hypervariable minisatellites in human DNA, previously Southern method for the two minisatellite probes 33.6 and 33.15 produced by hybridization.

Mother (M), the boy in question (X), his brother (B), sister (S11.Sl) and blood An 8 μg sample of blood DNA from an unrelated individual (U) was digested with Hinfl and ．． Electrophoresis was performed on a 7% agarose gel, and hybridization was performed using the Southern method against the probe. This led to the formation of a grid. The autoradiograph is shown in Figure 6. Mother's (M) DNA finger Fragments present in the crest are indicated by short horizontal lines. It doesn't exist in M, but it's not a problem. Paternal fragments present in at least one of the older siblings (B, Sl, Sl) are indicated by long lines. has been done. The "maternal" and paternal fragments transmitted to X are indicated by dots.

The DNA fingerprint of X contains no additional isolated fragments. Original taken with various irradiations All fragments were recorded from the autoradiograph. It cannot be recorded reliably, especially when playing games. The partially resolved and relatively weak band towards the bottom of the channel was ignored.

The first step was to establish the paternity of X from the pattern of hypervariable fragments. father was not available, but most of his DNA fingerprints are not a problem for the three people. Paternity present in at least one of the siblings (BS31, Sl) but absent in M It was possible to reconstruct it from isomeric DNA fragments. The 39 paternity cases identified in this way About half of the pieces were present in X's DNA fingerprint. DNA fragments are unrelated individuals Since it is rare that the DNA fingerprints of individuals (see individual U in Figure 6) are shared, , which very strongly suggests that X has the same father as B, Sl and Sl. . After excluding these paternally isomeric DNA fragments, 40 fragments remain in X, all of which All existed in M. This means that M is the mother of ] and Sl strongly prove that they are true brethren.

- A fragment in a person's DNA fingerprint is present in a randomly selected second individual It was already mentioned above that the average probability is about 0.2 for the Kitakou-0 Hupa people. father and The corresponding value for M is 0.26, indicating that the DNA index in these Ghanaians We determine that the pattern variability is not significantly different from that of the Kitaguchi-0 Hupa people. the below described In the probability estimation, it is assumed that all bands are shared with a uniform probability of 0.26. We make very conservative assumptions (quantified below).

The third question is whether or not X is related to this family.

X's r)N A fingerprint contains 61 recordable fragments, all of which are M and/or exists in the father. If X is unrelated, each of his bands The probability that they exist in their parents is 1-(1-0,26)"=0.45. Therefore, the probability that M and/or the father have all 61 of X's bands by chance is 0.4. 6X, where 5''=7X10-'', is clearly related to this family.

The next issue is whether a woman who is not M and has no curved edges can be X's mother. X The DNA fingerprint of a person contains 40 "maternal" fragments, of which we believe that about 25 are particularly He credits it as something he inherited from his mother. The remaining fragments are between the mother and father. shared and therefore cannot be used to give evidence about M's maternity , all 25 maternally isomeric fragments in X are present in M, L7. M is related to X However, the chance of sharing all 25 fragments is 0.26" = 2 x 10- ”.Therefore, X and M must be blood related and must be 2.

The final and most difficult problem is whether M's sister, who was not used in the analysis, could be X's mother. (The father, of course, must be M's husband). Band is 0. If shared with any person with an average probability of 26, then - person fragments also exist among fellows The corresponding probability is 0.62. M is the sister of X's true mother, and by chance C is The probability of containing all 25 maternal isomeric bands of X is therefore 0.62”=3 X10-'. Therefore, we do not have any room for reasonable doubt. Therefore, we conclude that M must be the true mother of X. Immigration authorities The authorities stopped the dispute against X, granted him residence in the UK and allowed him to remain with his family.

This difficult case is when 6 important family members with DNA fingerprints cannot be found. We show that even individuals can provide clear positive evidence of kinship. This case If X has the same father as his countryman,1 and the band that this father passed on only to The fact that (on average, 1/16th of the paternal band is transmitted) Basicized. Fragments unique to X, such as clearly weakens the light, but in fact does not necessarily invalidate the analysis, is unlikely to have more than 5 such paternal fragments in addition to the 25 maternal isomeric fragments. 30, if X and Ml are not related by blood, or if M is X's aunt. At least 25 (1ffi may coincidentally coincide with X and M) of a particular band of The potentials are 8×10-” and 9×10-’, respectively. Therefore, this analysis strong and affirming the kinship claimed in most such cases. or provide clear evidence to the contrary. Usually, of course, everyone involved in the family, For example, Yumiko's father C1 who is arguing about fathers! , where immigration makes it difficult for families to be reunited. If so, it may be available. DNA fingerprinting almost always solves these problems. can do.

-fingerprint p-number modification to pN~ There are 39 fragments uniquely inherited from the father, while M has 61 D. N A fragment was recorded. 1/8 of the father's heterozygous DNA fragments are B, Sl or not transmitted to S2, so the correction value for the number of paternally isomeric fragments is 39 X8/7=45. The total number of fragments in the DNA fingerprints of , M and father are approximately equal. Therefore, the number of fragments in M shared by the father is approximately (6m-45) = 16. . The average probability of band sharing in M and the father, X, is therefore 16/61 = 0. 26, based on a preliminary screening of a random sample of Northern Europeans. In agreement with the estimated value (x-0,2, control 2).

Approximately half of the 45 paternal bands are B, as expected for heterozygous bands. , Sl and s2 (18°24 and 18, respectively). M's band Is some of it also present in the father and thus transmitted to most or all of the children? Of the 61 bands of M, more than half are B, Sl and S, as predicted by Uke was inherited by 2 (32.38 and 39 pieces respectively). M's DNA fingerprint Shared band of n(j?tl) communicated to all children and communicated to half of children. n + Q if it contains (61-n) heterozygous bands.

5 (61-n) = 36.3, and if n = 12, 1 common to M and the father Consistent with the approximate value of 6 bands (see above).

The DNA fingerprint of X consists of 21 paternally isomeric fragments and 40 bands shared with M.

The proportion of the latter band that is maternal isomerism and not shared with the father is calculated in two ways. Ru. First, the number of maternal isomerism bands is approximately equal to the number of paternal isomerisms, 45/2 = It is 22.5. Second, n (about 12) of the 4o maternal bands in X are , a shared maternal/paternal band (see above), and The number of fragments that X specifically acquired from his mother is -2, approximately 25. .

I Σn Do-I1-92-Rall fixed rate Similar electrophoretic separation of the fragments in a second arbitrary person The average probability of matching by bands of variable and autoradiographic intensity is x ( x=0.2-0°26, see above) is defined as h. minisatellite fragment The larger the number, the lower the allele frequency probably and the better the electrophoretic separation. The frequency with which it is shared becomes less, so the fragment sharing probability X is non-uniform. Hatondo Since all t fragments are inherited independently, all n pieces in 4+ld people The highest probability that the fragment exists in any second individual is therefore xn. At X Inhomogeneity in the distribution reduces this probability.

U bribe, Tatorinotatsu! u1 car available If the shared bands always represent the same allele of the same hypervariable locus, X is related to the average allele frequency q by x"1q-q", which gives an individual The probability that a given band also exists in a compatriot is (4+5q-6q”+q’) /4(2-q).

Since X = 0.26, q is 0.14, which is shared by the record. The proportion of bands present in the first sibling is 0.62. This probability is The bands shared by the Slightly lower, assuming satellites have identically sized alleles .

Genetic Supervision Q Branch Office 1 animal Hiririie 9 miles Linkage analysis in humans, especially when disease loci are isolated together in large families To determine the possibility of using DNA fingerprints for the study of super-cIJ unusual DNA fragments. We investigated the DNA fingerprints of two large families (one isolated for a neurofibrotic mass) , the other is manifested by an autosomal dominant gene that is not linked to the β~globin gene population. (separated for genetic persistence of placental hemoglobin as measured by

Isamu 1 Nishi group's DNA 1'' which was around 1 away Blood DNA samples once digested with fl were run on a 0.7% agarose gel with a length of 35 cm. Electrophoresed and Southern method for minisatellite probes 33.6 and 33.15. to form a hybrid. Father (F) who is not affected by the disease, five sons (S) and her six daughters (D), whose DNA fingerprints are shown in Figure 7, were studied by the affected mother. I didn't use it. Offspring with multiple neurofibromas are indicated with a (+); The grandchild showed no signs of a neurofibrotic mass. Separated paternity (・) and maternity (0) Showing heterozygous DNA fragments, the original samples taken with short, medium and long-term irradiation Segregation into progeny was recorded directly from the autograph.

DNA fragments whose positions and relative intensities in each offspring match those of the parents only was recorded. A linked pair AB of DNA fragments that separates AB or -1 into progeny is a linked pair AB. connected by continuation lines. Alleles separating A- and -B are joined by dotted lines. has been done. A single maternal section showing evidence of ancillary linkage to a neurofibrotic mass. The fragment is indicated by an asterisk, and all six affected descendants will inherit this fragment and become affected. Four out of five children without this band do not have this band, and the genetics and nerve fibers of this band yielding a 10/11 agreement with the mass.

Forestry and mutual hojo Dangerous Emperor Fresh blood was diluted with an equal volume of 1x SSC (SSC, sodium citrate saline, 0.00% 15M NaC1, 15mM trisodium citrate, pH 7.0), Histobake (Histopaque-1077) (Sigma) and centrifuged. More nucleated cells were collected. Alternatively, thaw frozen blood in double volume of 1xssc. nucleated cells and nuclei were removed by centrifugation at 10,000g for 15 minutes. It has been made into a Jeffreys; A. J., (1979), Ce11, Earthworks. High molecular weight DNA was produced as described in Examples 1 to 10.

Southern method A 5 μg sample of human DNA was diluted with 20 units in the presence of 4 mM spermidine trichloride. Digested with HinfI for 2 hours at 37°C, extracted with phenol and precipitated with ethanol. Recovered by Mr.

The restriction digest was mixed with 16 μl of UZO + 4 μl of gel loading mixture (Ficoll 4001 2.5%, bromophenol blue 0.2%, tris acetate 0.2M, acetic acid Sodium 0.1MSEDT^1mM, p)l　8.3　'') and 5mg /nj! Ethidium bromide 2μ! Dissolve in horizontal agarose gel (Tris Acetate 40mM, sodium acetate 20mM, EDT^0.2mM, ethidium Mupromide 0.5. 0.7% Sigma (Si) in crg/ml (pH 8,3) gma) Type I agarose, thickness 0.7 cm x length 20 cm or 35 mark gel ) placed on top.

After 10 minutes of equilibration, all DNA fragments less than 1.5 kb in length were removed from the gel. Gels were electrophoresed at 2 V/am for 24-48 hours until run. Nitroce Produced on a lurose filter (Sart-OrlLIS % pore size 0.45 μm) The DNA was transferred by scanning. Human minisatellite M13 group Single-stranded probe DNA labeled with 32p from recombinant types 33.6 and 33.15 A was prepared and hybridized for Southern plotting in 1xssc at 65°C. cells and autoradiographed as described for tumor applications.

i2 Bunkou Kaji A BBC Model B microcomputer stores the separation data and chain analysis. Ta.

Table 2 summarizes minisatellite markings in families of neurofibrotic masses.

Table 3 father mother Probe residence, 6 33.15 Next day, 6 33.15 Number of recorded fragments (n) 24 17 16 16 Number of allele pairs (a) 3 3 2 4 Number of linkage pairs ( b＞　1　0. 1 0 Various loci recorded Number of (C) 20 14 13 12 The estimated total number of loci (N) 43 23 27 16 The number of different loci recorded (c) is , n-a-b. DNA fingerprints containing unseparated and therefore unrecorded fragments The whole is derived from N heterozygous loci (2N fragments). recorded (n-b) Assume that the apparent fragments of are a random sample of 2N bands in a DNA fingerprint. Then, the approximate total number N of hypervariable voices detected by a given probe is given by the following formula: Relates to the number of allele pairs by: (Margin below) Table 4: In families with nerve fiber masses (Separation of 5 hypervariable fragments) 47.7±2.7% To study the transmission frequency of super OJ fragments (Fig. 7), the exact Probes 33.6 and 33° out of n recorded and transmitted to one child in The number of fragments detected by The fragments present in the distribution must be from homozygous loci (ignored, which The maternal fragment that was not passed on to the child was 7, because the maternal DNA was not available. Could not record. The average transmission frequency (+3.2.M,) is also obtained.

Possibility of paternal or maternal fragments with initially excluded alleles and linked bands match on AB (AB or --) using all possible pairwise comparisons Linkage between the pair of fragments AB was determined by recording the number of progeny (i.e., the C locus was analyzed for each parent. See Table 3 to obtain a controlled comparison of c(c-1)/2) . Pairs of fragments appearing in groups of 0 or 11 children are allelic or closely related, respectively. Represents a chained pair.

By definition, a pair does not belong to any group; all of the C loci are linked to the observed distribution. Compare with the predicted value in case (U). In this case, exactly r(AB or -1 −) The number of pairwise comparisons giving descendants of the binomial distribution The loci are clustered and evenly spaced, and adjacent loci are separated by the recombination frequency θ. Compare the distribution with that expected if the Compare. Therefore, the group is (c-1)' Pffl flower rank [in the formula, A--1/2 I n (1-20)], the C locus (one or the other pair) (randomly sampled at the position of the gene), exactly r (A The number of pairwise comparisons that result in descendants of B or -1) is given by -9 below: Σ゛“′ [In the formula, X is the recombination fraction between two seats separated by the same distance i, and Xi is a temporary production function; x, −+/a (le −Z i A)].

spirit ”λ− The two minisatellite hybridization probes used in this study were I will explain in detail along the way. Probe 33.15 contains 29 repeats of a 16 bp variant of the core sequence. It consists of cloned human minisatellites consisting of In Prologue 33.6 The minisatellite repeat unit consists of the most conserved 11 bp 3' end species of the core sequence. Each trimer is repeated 18 times. The sequences of the core and core repeat units are shown below. As written: A Core GGAGGTGGGCAGGAGG33.15 AGAGGTGGGCA GGTGG33.16 AGGGCTGGAGG Due to differences in the sequence and repeat length of probes 33.6 and 33.15, , different types of long super-El odd minisatellite fragments in Hinfl digests of human DNA. This 4bp restriction endonuclease that will detect the pattern Long tandems in DNA fragments with ranking DNA-repetitive minisatellites Maximize the separation of various mini-sanarays by releasing .

nJ且拉t3 Kel D N A J! ＃　■qBunkaji Individual Mini Sanarai 1-DN To investigate the isolation of AltJi fragments, a large sibling group of 11 British individuals were treated with neurofibromas. The end of Reek (Reek 1 Inghausen Inn) An autosomal dominant disorder associated with tumors of the periphery and central nervous system was isolated. genetic markers The trait had not yet been linked to the disease.

l】Probes 33.6 and 33.1 in 6 affected and 5 unaffected children The blood DNA fingerprints detected in Figure 5 are compared with those of their unaffected father in Figure 7. compared. Separate the Minisanalite DNA1tJr piece into a 35cm long agarose plate. Maximized by gel electrophoresis.

Many of these hypervariable minisatellites, especially extremely large DNA fragments, have low It has a genetic frequency, and as mentioned above, it is rarely inherited by unrelated individuals. Therefore, many of these fragments in families with neurofibrotic masses exist in a heterozygous state. During its existence, only some of the older versions were passed down to later generations. Mother affected by disease 110) Obtain DNA Minisatellites derived from the mother even when not possible C3 was easily identified as a clear fragment. The paternity fragment is also similar. I was able to identify it. Using the lobes 33, 6 and 33.15, this 41 paternal DNA fragments and 32 maternal DNA fragments Recording the separation (Figure 7, Table 3), ``〉 was successfully completed. Although some fragments exist, they are either electrophoretically removed from the gel (β') or are not present. Completely separated! 11. Therefore, it could not be recorded in the rust.

This kind compatriot 1! All paternal or maternal DNA fragments By comparing the segregation pattern pairwise, allele pairs and phase differences of the fragments are determined. It is possible to identify pairs that exhibit a close linkage 4 in the state A (B in this sibling group B). The probability of simultaneous separation of a given pair of children is 1/1024)6 Paternity and Maternity Some examples of allelic pairs of fragments can be identified with both probes. (Figure 7). In addition, BL''-B33, 6 sends a chain pair of fragments to the mother and father. Detected. A similar linkage is seen in a second kindred (shown below), which - One hypervariable region hybridizes to 33°6 - 5i-! 1. Contains an internal cleavage site for fN and therefore cannot be cleaved and passed down the family tree. As a minisatellite “hablotype”, it does not separate at the same time and produces two or more fragments. This suggests that it is a long minisatellite/'satellite.

The polymorphic DNA fragment Y-recorded using whistle probe 33.15 is Therefore, it was not present in the combination of fragments detected. High on both probes Such fragments, which formed the prints, could not be immediately transmitted from the same parent to the same child. would have been detected as bands of equal size (i.e., 'chained'). Therefore, These two probes represent completely different woody subsections of human minisatellites. form a hybrid in the cut.

By eliminating alleles and linked fragments, each father and mother It was concluded that 34 and 25 distinct loci were recorded (Table 3). Approximately 80% of the , only one of the two alleles is isolated and the second allele is probably It exists in a poorly separated complex of relatively short minisatellite fragments. this This probably occurs due to unequal exchange in these tandem repeat regions. , meaning that large differences exist between the minisatellite allele pairs. Figure 7 Several allele pairs identified in , in fact, show substantial length differences. . Probes 33.6 and 33.1 were determined from the ratio of matching bands in alleles. The total number of heterozygous states present in the total DNA fingerprint detected by 5 is approximately 43 to It can be estimated that there are 66 children, of which about half are recorded by the father and mother. (Table 3). Measure the antagonism between the paternal and maternal fragments in this sibship It is not possible.

All recorded paternal loci are autosomal and can be either daughters (X-linked) or sons (Y-linked). does not indicate any special transmission to the chain). Furthermore, all pairs AB of paternal DNA fragments are Separate into descendants clearly and independently, with an average of the same number of (AB, --) or (A-1- B) Generate progeny*,,+tE, count the exact number, chain! -Predictions regarding the 7 constellations 2IQ distribution. N, sex DNA fragment showed similar behavior. More details Analyzing 1, the minimum locus spacing for these loci is ≦3 (lcM ( It turns out that the gap must be narrower than that, and at the same time segregated (linked in phase) or separated (linked in reciprocal state) with pseudoallele r and 17 (Table 4). Therefore, separable Minisanarai locus spans at least half of the 3000 eM long human genome - must be expanded, and therefore many or all of the human autosomes It must be lying down and scattered.

One maternal minisatellite fragment (Fig. 71) has a weak interphase linkage with the nerve fiber mass. 10/11 children agreed on this fragment and the disease. ing. (θ=10cM, p=0,006). However, 1.25 winnows Since the maternal loci were recorded, at least one allele of these loci However, outside of Il C;:,! U month! ,,,xRoh411-The observed degree of linkage in anti-deception The probability of showing is high (p = 0.24)ゎ case turtle Guang-Shifu family lineage qdany9 people, fingerprints-, 111817p ljImaichi 11D-possible- Function-η1-chain β-Mediterranean anemia and hereditary hyperfetal hemoglobinaemia (HPF) Analysis of DNA fingerprints on an extensive four-generation pedigree of Gujaratis segregating for H) spread out.

The autoradiograph of the label separation pattern shown in Figures 8A and 8B is shown below. It was manufactured in this way.

A 10 μg sample of blood DN A was once digested with -1 pfl, and probe 33.6 (A) or 33.15 (13) as shown in Figure 7). U2T I) N A fingerprint was produced. Relatively short (20cm) agarose gel Electrophoresis was performed. The relationship between ah is shown in Figure 9. In A, hypervariable fragment a, b, and C are closely linked and are all or all present in each individual in the family. do not. In B, bands g and IIl'Fll (H) separate simultaneously. individual ■7 and ■8 are rational twins and have indistinguishable DNA fingerprints.

Materials and methods were the same as in Example 8. "Simultaneous separation analysis in Figures 9A and 9B As shown in the figure.

In Figure 9A, 30 hypervariable fragments from ■4 and 27 fragments from ■5. Of, descendants! 111-11 on the possible linkage of the pair AB of the parental fragments. Screened.

Add further possible examples of chains showing at least 6/7 (AB, -m-) descendants. I was able to learn from blood relations. Two very clear examples of linkages are shown (a-f, disconnection in individuals). Presence of pieces a-f;・Fragments absent). Fragments a-C and e, f are completely simultaneous showing separation, fragment d has a tendency to separate at the same time as a-c, but sibling IV 1 -4 gives no information and 1.・-Rational twin ■7.8 has inherited a-c is a recombinant type that has d and does not have 7.

Figure 9B shows β-thalamic anemia traits (0, IF), H1'Fl ((l() and and the inheritance of minisatellite fragment g. Individual has 〉1% HhF (normal) or 〉3% 1bF (β-thalamic anemia trait), that (l/, one person has IIP Recorded as having FH. l　P　F　H and β-Mediterranean anemia 1flt syndrome The traits are III 1. -11 and ■5-8 independently separated 1-, -1 chain, Measured by Sagittarius. Fragment g II 11 F + in tested individuals 1 and complete 61 - separate at the same time.

expiration date As shown in Figure 9A and +3, the increase in II It is transmitted independently of quality and is not clearly linked to the β-globin gene complex. Measured by chromosomal dominant locus. A similar Sarcinian family is Gianni. Rayo-)teE? Reported in 1BOJ0.2.921-925 (1983) There is.

Figures 8A and 8B show 300 fragments and fragments of the grandfather (II 4). Twenty-seven fragments were recorded in the grandmother (■5).

Studies of their seven offspring (lull-11) have described a family of neurofibromatous tumors. Using the criteria listed above, these fragments have at least 22 distinct non-paternal origins. It was found to be derived from autosomal loci and 18 maternal autosomal loci. remaining DNs The A fragment proves an allelicity or linkage of other fragments, but in this small sibling group. It is not possible to prove (parental I) that a given pair of N A fragments is linked to a sib group of 7 (with a 1/64 chance of being transmitted by chance). Change Next, we looked for evidence of linkage in additional members of the family and found two examples of extremely strong linkages. It is shown in FIGS. 8 and 9. Probe 33.6? ::So゛C Detected fragment a, b and C are from ■4 to its child (Illll-11) and then grandchild (IV 18). ) is transmitted in a complete chain. The recombinant type is not found in the progeny that gives information on 14 people. (p = 4X 10-' for bands that separate at the same time), J = as shown This means that fragments a-C originate from one Choji locus, Minisatellite 1 “Hablo”. It means to represent Taibu. Also detected by BU1 Corps 33.15 Prove that band d is chained j7 to band a-C. However, one The other sib group (IV　l　-4) does not provide information because both parents have fragment d. , and the other (IV5-8) contains a recombinant type (-rational twin IV7.8) , the proof of linkage between band d and a-c population is therefore weak (θ-10eM, p=0.01), maternal bands e (detected by 33,6) and r (3 3, 15) are descendants of ■4 and ■5 and additional related sib group 11 115-20 and ■1 '7-22, no close linkage was shown (information on 20 people Progeny given, silk, no recombinants, θ-OcM, p=10-6). Probe 33. 6 and 33.15 detected various combinations of minisatellites, fragments e and f These fragments are two different autosomes because they do not cross-hybridize. Represents an example of reliable linkage between minisatellite loci. Finally, two linkage groups (fragment a -C and e-f) are not alleles at the same locus * (i!i person ■1 is a compound heterozygote with paternal a-C populations and maternal e-f pairs. collection of both The group was passed on to two of his four children, did not segregate as alleles, and Instead, it must be NM Rd from two unlinked hypervariable regions. Make sure that.

Maternal (■5) minisatellite fragments have significant linkage to β-Mediterranean anemia h1 trait therefore, it is closely linked to the β-globin gene cluster on chromosome 111. do not have. In contrast, the 8.6 kb long maternal fragment (g) & 1 progeny and grandchild It was isolated at the same time as old]F11 in three informative sibs BY (Fig. 9).

A tight linkage occurs between −]\zu(θ−OcM, p=2X10”4) and 12 progeny. However, 17 loci in 115 were investigated, and no KM recombinant type was observed. Despite the fact that this chain is still significant, <17 recorded Percentage of codfish in which at least one allele of the Ta locus coincidentally cosegregates with 1 FH. teeth,(). 004), and furthermore, the fragment g of ■5 is only one mini-satellite pair. Hi oN A fingers of all individuals shown in Figure 9 digested with pu3A instead of nflO This was investigated by examining the crest. All aggressive fingerprints are Corresponding 5 of similar size to fragment g predicted for telite fragments. It contained the aq3A fragment (8.2 kb vs. 8.6 kb).

Nosawa Human genealogy analysis is mini-satellite fragment 1! - DNA fingers detected by Even in families where one or the other parent is not available for research, there are many variants. Zygosity 1) Demonstrates that it can be reliably used to study the separation of fragments into N. This way Using two probes, one person can simultaneously test up to 34 super II probes. J variable locus can be analyzed, and in human genetics, including RFLP, It is possible to analyze genetically marked trait production rates that are much higher than those obtained with methods. . Various minizaterra (1 = stable remains of fragments) together with low frequency of individual fragments As shown in the chain example found in the family tree analyzed in 2.'), These fragments become ideally suited for linkage analysis. These super-cool mini satellites could be an x, 11 replacement hotspot, but the failure that occurs with long minisatellites An outline of equal exchange 'J: J-speed (approximately 0.001 per 1 exchange) is a mini-satellite It is not enough to significantly disrupt the link between the locus and the neighboring path, e.g. the locus of disease. stomach.

Ultrasonic waves detected together by minisatellite probes 33.6 and 33.15 The total number of abutments is calculated to be approximately 60. Two alleles for about half of these loci At least one of the children (1i1) can be separated by a given DNA fingerprint and therefore Therefore, if the DNA fingerprints are different, the spectra of the loci tested will not be identical.

Most or all of these loci are genetically linked and, therefore, are present in the human genome. The scattering must be 1.7 over a significant proportion of the system. their exact location Since the location is unknown, cloning and individual hypervariable minisatellite loci We have to wait for regional positioning. (Oddly enough, in both families studied) , but no minisatellites have yet been observed on the X or Y chromosomes. Approximately 43 A different locus is found in the IPFB family (along with the flit family 4). Possible physical traits in the family's fathers were recorded. The X and Y chromosomes together are male There are 43 randomly distributed loci on these chromosomes, making up approximately 5% of the genome. The probability that it does not exist is (0,95)” = 0.1. Therefore, the somatic minisa The apparent loss of telite is not significant.

These dispersed hypervariable minisatellite loci are linked to neurofibrotic masses and HPFH. As shown by the hypothetical example of a chain, the link to the locus of disease and the label 0) Research G, 2 Q (legitimate child) 1, (if', it is different from the next tp locus genetic analysis, mini, The true allele is the name-family 6, ', 13 diseased deer and II i! !　 ! - I don't think it's okay, so it's a fl between small unrelated families! : % chain de ...I can't bring myself to see the evening.

Instead, to 1) only IA fingerprints are suitable for a wide range of pedigrees, C and the most rational, target It is especially suitable for studying fast chains of dominant disorders in sib groups with large populations. is 31 , up to two points, 1" - F (published; (, 6 and 3:3.15, 4181 people C, In 7, write 2 letters of Tsuneishiro A, Do υ'-'i + J Hanko, up to 34, 2 letters: ! 7. OJ ability is closely linked to the locus of a given disease in which at least one of the ,”3,3000cM,I・l! l　gA　pl　,74,の3　=' Assuming an arbitrary variance of t'j-'fy, it is 20%. ,, 11 yen; 1 (For a family with an extensive lineage of l-eel, there is only one pair of one leg in the constellation of Tondo. Since the gene is recorded -), this probability is about 10? (to detect chaining becomes , this allele must be linked in phase to the disease locus. child The probability of these is 50%')')y,,,b,L is 7,-. One large brotherhood? Record of 7 and more than 104 supermutable constellations and extensive family pedigrees. It is necessary to have a record of more than 208 constellations. 1-Also exceeds the total number of Gins detected by the two satellite probes.

However, probes 33.6 and 33.15 are essentially Overall and 1. 1 to detect different combinations of human This suggests that the total number of minisatellites is large.

In conventional IIZ)/pedigree analysis that uses limited single-locate markers,! ! wisdom and Demonstration of linkage between a disease locus is usually directly based on the approximate genomic location of the disease gene. 6DN, which can be further established by giving a location and analyzing additional pedigrees. The reverse is also true for A fingerprints. Possible link between super 6J unusual DNA fragments and disease Further analysis of the strands involves fragmentation by preparative gel electrophoresis and cloning. Isolated and possible. Directly clip the minisatellite from the isolated minisatellite. Either use specific sequence DNA segments that Establishing a locus isomeric hybridization probe by using Nisatellites can be measured. These locus isomer probes can provide additional information on linkage data. used to expand into the human genome and to localize minisatellites within the human genome. be able to. This ° approach clearly shows that in Gujarati ancestry, 11 Clone 8, 2 and 1 Shu 3A minisatellite fragments linked to PFH This has now been confirmed by testing.

As mentioned above, each human minisatellite contains a different variant of the core sequence. The probes 33°5, 33.6 and 33.15 have different DNA fingerprints. , and therefore hypervariable minisatellites in humans and other vertebrates. Detect various combinations of regions. In particular, probes 33o6 and 33. '15 may result from differences in the length and exact sequence of the repeat cores present in each probe. to detect most or all of the various combinations of minisatellites (third application and 8, J., Jeffreys, V., Wilson, S.L., Thejn%. “DN” by D, J, Weatherall & B, A, J, Ponder A. “Fingerprints and linkage analysis in human genealogies”) Other: To test the ability to detect additional super-01 variable regions using A series of synthetic minisatellites were produced.

Ikri-j　,,-(t- Person ['-Minnie Satellite upper-02 synthesis and black-Ninri 1 multi-core pro- - The approach to manufacturing the tube is outlined in Figure 10. Figure 1 () is E, 7li (E , col i) transfer!・Initiator array (Ch i, G CTGGTG It is. The process of making poly-Chi 1 copy uses standard DNA techniques. 1. The following I: is: 1. Length! (Synthetic oligonucleotide containing tandem repeats of the Cbi sequence of Nuk1/Odyo-do) Il, W, D, Matt, et al. (1984) (■2.) I I (OJ,, -1 branch, 801-805) and G, Brenner & W, ν, by the method of Shaw (1985) EMBOJ, 4.561-568. Manufactured by The first oligonucleotide consisting of a duplex of ChiO complementary sequences is Synthetic Rimata.

2. 10mM MgC1□, 10m DN 5. The sequence of the second oligonucleotide that formed the short double-stranded segment of A is head-to-tail. Anneal and molecule with a 5' overhang of the appropriate 3-nucleotide length for ligation. chosen to occur.

3. Phosphate the 5' end using T4 polynucleotide 1/otidekinase and ATI' It became.

4. A; - Use T4 DNA ligase and ATI' to remove the ligated DNA fragment. Repeated Chi polymers were prepared by linking them together with a gull-like attribute.

5. Separate the linked polymers by size by electrophoresis on a 1.5% agarose gel. Polymers larger than 15 (l base pairs in length) were separated and subjected to electrophoresis on DE810 paper. (+) retzen isolated from.

G, , Be11ard, M, %5assone-Corri+P, Fill-in repair using Flenow fragments of & Chambor+, p,, ■ From the end of the plant, J3mp19 RFD N A (J, Messin G & J, Vieira, 1982, Gene IL, 269-276 ) into the Sma I site.

Transformed into the ligated DN A ;ji, . Single-stranded phage DNA was isolated from each white plaque.

7° DNA inserts of each cloned isolate were isolated using the dideoxynucleotide method (M, + 3. Old ggin, T, J, Gibon & H, F, llongs 1983, Proc, Nat, Acad, Sci, t! SA80.39 63-3965) to determine the length, direction and alignment of the polymer inserts. It was measured. 5 of the Chi sequences oriented 5'→3' in the mature-terminal phage DNA. A recombinant M13 phage containing 12 repeats of 0 was found and was transformed into M13 co It is called A [i.e., the inserted part is poly(Chi), and the complement of poly(Chi) isn't it〕.

8,3J)-labeled single-stranded hybridization probe was added to phages 33°6 and 3 3. Primer extension using the same method used to prepare the probe from 15. Manufactured by the compression method.

Example −↓−↓2π and −5− )-” qEnter history-ρ4-2 catty←()Person-Amy-Soichi-Specialist-Tailor-ite, , 0) 9-j1 Using the technique described in Section 1-, further five types of core arrays were synthesized and 7 , M]: 3 was cloned with the multi-core combination I cost type and 1.2, and the recombinant type M 1 :3.-1aA-F was obtained. Core modification to change core length and exact alignment I chose a variant. Table 1 shows the repetitive sequences in clone M13 core A-F. 6 commonly used probes 33o5.33 described with core sequences and early applications ．． 6 and 33.15. Highly variable in the core sequence Attach a 1- line to the base. The orientation of each insertion part in Hector M13mp19 is also shown [- ..., the insertion part is poly(core).・- means the insertion part is poly (:J, ) indicates a complementary sequence. ] The base marked with a lowercase letter 62 is from the 217 sequence. indicates the departure of Summarize the unique characteristics of each array in “annotations”.

(Margin below) Example 16 In the initial screening, DNA digests from two unrelated individuals were tested at 33.1 5 and probe M13/cores A to F, respectively. 2 An 8 pg (7) sample from an unrelated placenta (1°2) to (DDN) was incubated with HinfI. Digested with Hybrids were generated for each 32p-labeled probe by the procedure described in the section below. formed. The resulting autoradiograph is shown in FIG. All probes are individually A large number of DNA fragments were detected in humans.

Expanded rice Probes B, C and D each probe DNA segments that differ substantially between two individuals. A fingerprint was detected on the piece. Fingerprints vary depending on the probe, but some fragments may be one selected by a larger number of probes and to some extent by probe 33.15 overlapped with the combinations of hypervariable fragments detected. Probes B-D are all DN A.Suitable for fingerprinting and testing in humans and other vertebrates. We conclude that this expands the number of hypervariable minisatellites that can be made. Here, the core array Effective fingers obtained using core C containing only the central most conserved 7 base pair segment Crests are important.

Further truncation of the tip of the core to generate six base pair repeats in core E results in DNA Fingerprint Bakun was completely changed and almost shared by the two individuals tested. (These fragments also show extreme polymorphic changes.) (No) Therefore, Core E is compatible with previously used probes for DNA fingerprint analysis. 33.5.33,6 and 33.15 are unlikely to be as useful probes. do not have. This is also valid for -m, which means that for NA fingerprinting there is actually a minimum of 7 bases. Suggests that paired core sequences are required. Also, inside the core (M2S/Core G) Disruption of the most conserved regions of the mind reduces the complexity and variability of DNA fingerprint patterns. It seems to be that.

M2S core A (polyChi) is a novel and strong hybridization NA fragment. This results in a strange pattern. Many of these fragments are extremely large (>15kb) , containing the less isolated and sometimes present Hinfl cleavage site can be derived from long satellite sequences of

in a neurofibrotic mass that was extensively characterized using probes 33.6 and 33.15. We further analyzed the patterns produced by M2S Core A in affected families ( (See Example 8 and Figure 7). Figure 12 shows a father (F), six daughters (D), and five Figure 2 shows a DNA fingerprint from a Hinfl digest of DNA from the son (S) of . Permanently dyed Individuals affected by neurofibromas, which is a dominantly inherited cancer, are indicated by 10. A sick mother? Their DNA was not available. Indicates separate paternal (・) and maternal (○) bands. vinegar. Bands connected by solid lines are linked, bands connected by dotted lines are alleles. Separate as. The band with (x) is pre-defined using 33°15 was detected.

J, l′i fruit Unlike the DNA fingerprints obtained using B1 Cope 33.6 and 33.15, , the level of variability is relatively low, and a large number of lvi fragments are transmitted to all descendants. (Immediate - Jul, 2, these cuts) 1 is the 45th group 1. Second, general information. , J Kei 1. rather than 1-2 cases, they often exist in a homozygous state). 6 heterozygotes Placement of the sexual paternal band and 9 heterozygous maternal bands (but 11 children's siblings recorded in groups.

Test the allele pair of one of the paternal bands and the maternal band with probe 33.15. I put it out. After eliminating allelic pairs and concatenated pairs of bands, two new (, Probe 7 paternal and 6 new maternal f loci 33.6 or 33. L remains unrecorded in 15, paternity loci of 34 @ previously recorded and 2 Compare with 5 maternal loci. Therefore, polyChi probes are useful for human genetic Has limited use in analysis.

The results of Examples 16 and 17 are underlined in the last column of Table 5 and discussed under the main application. Confirm the importance of the nine dominant nucleotides. These are also valid professional The main purpose of the core is that it requires a minimum sequence of 6 nucleotides. It is very useful in confirming predictions made. Therefore, core E has the minimum availability 2 and other sequences of 6 nucleotides, such as the sequence TGGGCA discussed below. can show marginally improved utility. Increase to 7 nucleotides is extremely dramatic within the framework of the investigation.

In an attempt to define the essential elements of a useful core sequence, alternative nucleotides The variants X and Y used above to show that can be given: not X=A or CP=G y=c or T (not Q=A) W=A or T (not R=C) V=C or G (not S=T) (〇－Optional) () = Not used in the discussion below.

Using this terminology, aspects of the invention include the following repetitive core sequences: GPGGGCmi GGWXG (6) can be said to include polynucleotides containing.

The above sequence, of course, represents the most representative 12 nucleotide sequence. death However, the 7-nucleotide core C was also found to have significant utility, and the above To include those with “allowed” variants from the 12 core sequences of The embodiment is the following 7-core array: PGGG (JG (7) It can also be said to include polynucleotides containing repeats of.

Of course, P is preferably equal to T as in core C. other most preferred A suitable number would be eight.

For clarity, the homology percentages have already been shown in Table 3. People nib for this purpose For lobes, the iterations are exact iterations of the minisatellite as a whole. Homology can be shown by core sequence homology. The above-mentioned B1 Corps 33.6.33.15 and 33.5 shown in parentheses are not required. is also not true. This is due to mutations that occur between repeats.

Core F probably shows the lowest homology rate in terms of utility. However, this discrepancy This is probably due to the central group present in the core: TGGGCA (8) exaggerated by the destruction of Among the probes B, C and Y) in which the above groups are the most effective Probes A, E, and I, which are present in the It is worth noting that. Therefore, at least 70% of all phases that satisfy equation (6) It is expected that more effective probes with similar rates will be obtained.

Formula (8): %formula%) It is noteworthy that the center 6 Boris Kreotide of is also destroyed in Core E.

Presumably, the six nucleotide core sequence described above would be more effective, but the length could be reduced by nucleotides. It is expected that increasing from 6 to 7 will demonstrate even more dominant characteristics.

Accordingly, embodiments of the invention provide polynucleotides comprising repeats of the core sequence TGGGCA. It can be said that it contains chido.

More generally, the invention provides that - in an embodiment, the sequence TGG is present in every repeat sequence. It is said to include a polynucleotide according to the above "first" definition in which GCA is present. I can do it.

Viewed from another aspect, the present invention provides that read with the same 5'-3' meaning; at least 6 consecutive nucleotides It is said that it includes a modification of the above-mentioned “first” definition of polyscleotide, which represents a sequence of I can do it. The “core” is defined by each iteration as long as all units contain the sequence TGGGCA They do not necessarily have the same sequence at the ij position.

The remaining groups in each unit are represented by formula (6) (or more preferably 2) within the constraints of the total unit length. has at least 70% homology with the sequence of (2)).

Poetry of Hada Twins Fufu Sex ■ Anatomy - Measurement of zygotic fertility in twins is useful for epidemiological, genetic and obstetric studies. This is important because there is a difference in the predictions of rationality and fraternal twins. − Ozygotic twins have a lower birth rate, lower weight, and more medical complications than fraternal twins. The mortality rate is high. In the Caucasians, about 30% of newborns have different sexes, and therefore They are dizygotic. When the placental membranes are tested, another 20% of cases are monochorionic; They are always found to be identical. The remaining 50% are relatively unique in the group. of a fixed proportion, of the same sex, with a dichorionic diamniotic placenta, - rational or dizygotic It can be. To determine zygotic fertility in these cases, general appearance evaluation, finger A variety of methods are used, including staining, skin grafts, taste testing, and measurement of genetic marker traits. It was done. The latter is the most reliable, with an accuracy of 95-98%. However, most Because the average heterozygosity of protein and antigenic variants is relatively low, such Many marker traits must be investigated.

In the example below, DNA from 12M1 newborn twins is transferred to a minisatellite as described above. The test was carried out using a native DNA probe.

The obtained DN A “fingerprint” varies from person to person, and only ovoid twins are identical. Prove that there are no negative or negative effects. Determine zygotic fertility from sexual observation or placenta μ; In the 7 cases that could be determined, DNA results were consistent with these findings.

In the other five twin pairs and two triplets, DNA analysis determined zygosity. It was possible to measure quickly. Therefore, the DNA probe according to the present invention In all cases of pregnancy, zygotic fertility can be positively measured. 12 using the 31 end-strand DNA probes 33.6 and 33.15. The zygosity of newborn silk twins was measured and determined. The details are shown in Table 6.

Table 6 Case 4 Fetal age, gender, placenta accreta DNA bar-11~-Sho-he-1-7--, Tanis - 138 weeks FF monochorionic same 238 weeks MF dichorionic non-identical - 330 weeks MM dichorionic identical 433 weeks FF monochorionic same 540 weeks FF dichorionic non-identical - 637 weeks MF dichorionic non-identical 732 weeks FF dichorionic same 834 weeks MF dichorionic non-identical - 939 weeks MM dichorionic non-identical 1038 weeks MF dichorionic non-same - 1127 weeks MM dichorionic non-same 11,235 weeks FM dichorionic non-iso-*All placentas are diamniotic. Ta.

A sample of adult blood collected at the time of birth or a sample of peripheral blood obtained from each infant the day after birth. (0.5-1.0 ms was used for DNA extraction. The placenta was tested and the placenta was Mono- or di-amniotic and mono- or dichorionic were determined. DNA was extracted using standard methods (O ld.

J, M,, 111gg5. D, R, author, genetic analysis, D, J, Wheat herall, ed., The Thalassacmias, in hematology Extracted by Churchill Livingstone, 1.983), 110-1 5p was first digested with 1nfI. Transfer the sample to a 0.6% agarose gel with a length of 22c+m. At 45 V for about 36 hours, all DNA fragments less than 1.5 kb in length were electrophoresed. The gel was electrophoresed until it was removed from the gel. Filter DNA through nitrocellulose filter - by plotting and baked under vacuum at 80°C for 2 hours. . - Label the end-strand DNA probes, 33.15 and 33.6, with 32p as above. I did it.

The results are shown in FIG.

In Figure 13, rows l and 2 use a 33°6 single stack minisatellite probe. shows the DNA band pattern obtained for each twin of case 1. Example 3~ 19 shows the "fingerprint" obtained using one tM33.15 probe, case 1 case 2 is in columns 5 and 6, case 3 is in columns 7 and 8, case 4 is in columns 3 and 4, case 2 is in columns 5 and 6, case 3 is in columns 7 and 8, case 5 is in columns 11 and 12, case 6 is in columns 13 and 14 Case 7 is not shown in columns 15 and 16. Columns 17-19 are for women, men and women. Showing three triplets. Comparing rows 1 and 2 with rows 3 and 4, two types of probe 3 3.6 and 33.15 to detect various combinations of minisatellite bands. I understand. Size labels are given in kilobases.

In seven cases (see Table 6), the sex of the twins and examination of their placental membranes revealed that the nodes were isolated. We were able to measure zygotic fecundity.

All twins with 1f monochorionic (or monoamniotic) placentas (e.g. case 1) Although it is rational, - about 50% of ovoid twins have a monochorionic placenta (2). It wasn't too much. Therefore, the twins in case 1 must be -rational and 3 3.15 and 33.6 probes showed the same DNA pattern (Figure 1) In 5 cases, the twins were of different sex, 33.15 and 33.6 Different lobes showed different band patterns. Case 3.5.7.9 and 11 Twins are of the same sex, have a dichorionic placenta, and are rational or dizygotic. sell. DNA analysis revealed that the 2 & Il twins (cases 5 and 9) had different bandpass patterns. turn and were therefore dizygotic, but in the other three pairs (cases 3.7 and 1]) It was found that they had the same band pattern, indicating -reason.

Two sets of newborn triplets were similarly studied. In both cases, the mother induces pregnancy I was taking fertility drugs. Each triplet showed a unique bunting pattern (e.g. Figure 13, columns 17-19).

The results are shown in FIG. In FIG. 14, columns 1 and 2 have one line 13433.1 Columns 3 and 4 show the results using duplex 33.15.

Nukiage Ni Single-stranded or double-stranded probes are suitable for each diagnosis, but many Figure 14 shows that a large number of bands can be distinguished using radioactive end-strand probes. ).

As an alternative to these -terminated probes, they are becoming more popular in most laboratories. We investigated the possibility of using corresponding double-stranded probes. Double-stranded probe used i) Two ti600bp containing “core” minisatellites from λ33.15 Pst I-Aham fragment and 1i) “core” minisatellite from 233.6 There were two t4'720bp Haem fragments containing These are DNA 0.5-1. OX until 10”cpmO specific activity is reached. Labeled by translation. Pre-hybridization and hybrid forms The formation conditions were: i xssc and 10 % dextran sulfate was used as described in the standard method above. .

Filters were washed for 1 hour in 1x ssc at 65°C and filtered using an intensifying screen. Electrophoresis was performed at 70°C for 1 to 3 days.

Plate earthworks and superior study In the seven cases in which zygotic fertility could be measured independently, the results of DNA The results were consistent with the conclusions based on sexual observation, observation, and placental testing. In the other five groups, Unambiguous measurements of zygotic fertility were possible using minisatellite probes. similar The two sets of triplets studied were found to be dizygotic.

Zygotic reproduction of twins depending on sex difference (dizygotic) or monochorionic placenta (-rational) In 50% of cases where sex cannot be determined, genetic analysis must be used to determine LJ. stomach. The amount of information provided by such a test depends on the degree of versatility at the loci tested. and the number of loci tested. Based on measurements of numerous red blood cell antigens and enzymes, A zygotic fecundity of around 95-98% can only be achieved if the associated allele frequencies are known. Accuracy can be achieved (Nylarider, P, P, S, Author, Twin Birth Phenomenon, Barron, S.

Lo, Thomson+^, M, Pffi, 0bst, etical Epi demiology, London, Academic Press, 1983 : 143-165) *Similarly, many studies using several different DNA probes Analyzing restriction enzyme site polymorphisms in occur in percentages (Derom, C2, Bakker, E,, Vli et-ineck, R, Derom, R, Van der Berghe , H., Th1ery, M., %Pearson, P., Using DNA Variants Zygogeny measurement of newborn twins, J. Med. Genet. , 1985. 228279-282), the minisatellite probe according to the present invention solves this problem. overcoming the problem. This is because the number of hypervariable DNA segments to be detected is large and This is because it can change rapidly. As above, hybridize minisatellite fragments are rarely shared among randomly selected individuals. two unrelated people Probes 33.6 and 33.1 for individuals to detect various combinations of hyperconvex loci It has already been shown that the probability of showing the same DNA fingerprint in 5 is therefore astronomical. (p<<<No-”, see Example 4). Among siblings who share about half of the fragments The probability of such a “false positive” diagnosis of genetic identity is <10−”. be. Therefore, we assembled -terminal hybridization probes 33.6 and 33.15. When used together, they provide greater accuracy than traditional genetic tests.

−Hybridizes to some of the relatively weak bands detected by the end-strand probe. Even using more common double-stranded minisatellite probes that do not form Figure 14), lowering the “false monozygotic” rate from DNA fingerprints to <10-’ can obtain sufficient information.

Another advantage of this zygotic fecundity assay is that only a very small sample is required for analysis. That's true. Usually half a meter! ! Sufficient NA can be obtained with peripheral blood or prepared blood. Ta. Measurement of zygosity in newborns and older twins and triplets By using this clear method, we can determine the determinants of different types of hyperlipidic pregnancies. and more accurate epidemiological studies on the effects.

In the example below, the molecular weight label is not obtained in the autoradiograph, but is included in the other results. As mentioned above, the general range of validity was 1.5-20 kb.

DNA 5 to S DNA fingerprints detected by minisatellite probes 33.6 and 33.15 The individual specificity of DNA fingerprints makes them ideally suited for identification of individuals in forensic medicine. Become. The only uncertainty is that DNA may be present in e.g. dried blood or semen. The question is whether it can survive in a sufficiently undegraded state for DNA fingerprint analysis. Forensic medical examination To measure the analyzability of the body, Ilome OfficeCentral R e5earch Establishment, Aldermaston P Preliminary studies were conducted on D□NA samples provided by Dr. eterGill. did.

fl 岨 The dried blood and semen Q traces on the cloth were analyzed by DNA extraction performed by Dr. G111. extraction (dissolved in SO5 in the presence of IMDTT, then extracted with phenol and extracted with ethanol) The mixture was allowed to stand at room temperature for various times before being precipitated in a vacuum chamber. Similarly, fresh hair roots and DNA was extracted from vaginal swabs taken before and after sexual intercourse. DNA sample with HinfI Digested with plotted above. Filters are labeled with ``tp-label'' using the standard techniques of the invention described above. It was hybridized with the single-stranded real lobes 335.

The samples electrophoresed were as follows: 1. Semen herpes 40 μm on cloth; 4 The one after a week.

2. 40μl of fresh semen.

3. Fresh blood-fi60μm0 4. Blood trace on cloth, 60 μN, after 4 weeks.

5. Blood sequence 6oμr on cloth, 2 years later.

6. 15 hair roots.

Note: 1-6 were collected from the same person.

76 Cloth' 2 blood eczema 60ttl, male, fresh.

8.7 Vaginal swab taken from G11 1 hour after intercourse.

9. Same as 8 except that the sample was collected 7 hours after intercourse.

Vaginal swab taken from 10.11.

11. Blood rash on cloth 60p12. Ladies, fresh.

The obtained DNA fingerprints are shown in Figure 15, and each sample had a sufficient NA ( About 0.5-3 μg) was extracted. DNA can be detected even after 2 years in dry blood herpes. Until 2 months have passed, dry semen does not decompose very much. 1. same It produced a DN A fingerprint identical to that obtained from an individual's fresh blood and semen.

Similarly, N A fingerprints can be obtained from as small as 15 hair roots. Ta.

Vaginal swabs taken after sex also produced undegraded DNA fingerprints. But long 2, the swab pattern is primarily for female blood rather than male blood. Agreed. This means that most of the DNA collected from swabs is collected using cotton swabs. It was confirmed that the product was from bidermal cells in the vagina that had been exfoliated during the period of treatment, and not from semen. and Nevertheless, in the postcoital sample, three additional female A band that does not belong to the band was detected. These are the main bands in a man's blood. ・It must match and be Hl-derived from semen. A band like this, The results of these preliminary experiments indicate that DNA 1. are maintained intact in various forensic specimens for identification purposes. :)　N　A　J’j□゛Scale 1 and

For example, it is possible to positively identify a rapist based on the emotional scars on the clothes of an elderly shopkeeper. strength When using vaginal swabs from rape victims.

It is not very certain because it involves vaginal DNA. However, semen D N AO single; ■(7) Requires semen 2-DTT or mercabutoecinol ~ compensation before mediated reduction. When this woman's DNA is removed by 1s melting, an NA fingerprint becomes even more obvious. Jiru. In this case, the identification of the rapist may be based on the herpetal fluid and/or the DNA finger of the vaginal swab. This is possible through pattern analysis.

Subsequently, in recent research conducted in collaboration with Dr. G111 - the above-mentioned and preliminary Using a separation technique, clear "fingerprints" of semen DNA can be obtained from post-coital vaginal swabs. It was confirmed that

Figure 15A shows DNA fingerprints ( Column 3) is shown. Column l shows the fingerprints from the other man's blood sample, and column 2 shows the fingerprints from the other man's blood sample. No DNA fingerprints were found from the blood obtained from the woman. Female cell nuclei from swab, SD S/protiJ~-ase1 (preferentially lysed by preincubation in the mixture).

Semen nuclei are not affected by this process and can therefore be separated from the female component by centrifugation. can be released. After that, the semen nucleus was mixed with SOS/proteinase/DTT. It was dissolved by treatment with a compound.

It is clear that this separation operation was completely effective.

Semen DNA fingerprints from semen-contaminated vaginal swabs are obtained from the other man's blood. The fingerprint was a perfect match.

``Human Mini Satellite'' 2 L ships a month (11i experience, luck Jμd Maru's first time) 9J Sugen A! Crest.

``剌'''' Knee-25 DNA samples were prepared from blood taken from dogs, cats, sheep, pigs, crows, and cows. 8μg A sample of The restriction digest was recovered by ethanol precipitation after phenol extraction.

The restriction digest was redissolved in water, electrophoresed on a 0.7% agarose gel, denatured, Transfer to a 5chleicher-Schuell nitrocellulose filter. , '32P-labeled human minisatellite probe 33.6 or 33 as described above. ．． 15 was used to form a hybrid.

□Dog A DNA fingerprint obtained from a family of dogs using probe 33.6 is shown in FIG.

The samples electrophoresed were as follows: Row 1 Beagle-sized father Row 2 Graybound's mother Examples 3.4 and 5 “Greagle” puppies of these parents ( female, male, male respectively).

A complex and detailed NA fingerprint similar to that obtained from human DNA can be obtained from each person. It was. By comparing the patterns obtained from fathers and mothers (column 1.2) As can be seen, the DNA fingerprint showed significant variability. All three puppies are and all bands that can be traced back to the father and/or mother. It has a DNA fingerprint. Therefore, these DNA fingerprints, just like in humans, Useful for paternity testing and pedigree analysis in dogs.

The DNA in samples 1-3 was slightly degraded, which caused the largest (slowest swimming) (moving) band intensity was preferentially reduced. Despite this disassembly, from parents Transmission of the band to offspring was easily measured.

Purchase plate or fork FIG. 17 shows that using (left) probe 33.6 and (right) probe 33.15, Showing DNA fingerprints from a family of short-haired domestic cats.

Column 1 Mother Column 2 Father Row 3 Koneko Probes 33.6 and 33.15 produce informative DNA fingerprints. Koneko no Ho Most bands can be recorded as maternal or paternal and therefore these DNA fingerprints are suitable for pedigree testing as well as individual identification in cats.

tLL d D obtained from various sheep using probes 33.6 (left) and 33.15 (right) The NA fingerprint is shown in FIG. The samples were: Row 1 Cross-bred female Row 2 Cross-bred females Row 3 Dorset female Row 4: Hampshire x Dorse Soto female Row 5: Dorcent male Both probes yielded individual-specific DNA fingerprints from winter grass. DNA fingerprint , weaker and less complex than the human DNA fingerprint, but useful for identification and genetic purposes. It is still useful for Probe 33.6, in addition to the weak band range, detect one or two very strong polymorphic bands. These bands are probably , from a single minisatellite locus showing extremely high probe 33.6 homology. It was induced.

■１１　Gogyu Guyu Figure 19 shows three different pigs (columns 1-3) and three different horses (columns 4-6) [sex Separation and breeding show DNA fingerprints from [unidentified]. probe 33.6 and 33.15 produce species- and individual-specific DNA fingerprints. Especially probe 33. 15 DNA fingerprints are weak and extremely rare compared to the corresponding human DNA fingerprints. However, the combination of both probes is still useful for individual identification.

le l cow Figure 20 was obtained using probe 33.6 for the cow family and additional cows. The DNA fingerprint of the base 1nfl DNA digest is shown.

The samples are as follows: [Column 1 Human] Row 2 Inside the company Row 3 Father cow Calves in rows 41 and 2 Row 5 Angus bull Row 6 Flesian benthic As in the case of sheep, pigs and horses, it is quite a front wheel, but still individuality) 1 sex D NA fingerprints were obtained from each animal. In calves, all bands are internal and/or We were able to trace the origin to the father's cow and confirm the animal's pedigree.

Figure 21 shows the results using probe 33.15 on the same maggot DNA.

The individual columns indicate:

3. 1 and 2 calves 4° Angus bull 5°Fresh)'n Beef hybridization that produces strong, inseparable, and complex patterns of NA bands. arise. In the BJLLII digest, this strong signal was mainly due to extremely This signal is restricted to large DNA fragments, such as tufted sequences or regions, perhaps This suggests that the DNA was derived from a previous satellite DNA.

Brief autoradiography of the base 1nfl digest revealed 45-50 base pairs. towards the bottom of the gel, with periodicity between the “rungs” between (data not shown). ・Shows a “ladder” shaped band. Therefore, a strong signal probably has a length of 45-5 It is derived from a satellite with a repeating unit of 0 base pairs. strong signa in bovine DNA digested with PvuTi, Danpei1 or AluI. The satellite DNA repeat units are severely disrupted and these restriction endonuclei contains one or more sites for each of the Hinfi or BLL This suggests that it does not contain a site for II. These are exactly 46 base pair repeats 1,720 bovine satellites (E, Po5c) consisting of 100,000 tandem repeats of the unit hl and R,E, 5treek, 'Cow 1.720 Satellite DNA Pro Type Arrangement”, J, Mo1. Biol, Dish, 147-153: 198 0).

Sequence of 1.720 repeat units and core probe 33.6 repeat units and 33.155 repeat units A comparison with the recursive unit is shown below; Core GGAGGTGGGCAGGAXG 11 ha　DdeN33.15　-　8GAGGTGGGGCAGGTGG　IA LLL　133.6　8GGGCTGGAGG　Pvu　II It is clear from the above formula As expected, the 1.720 satellite repeat unit covers almost the entire 3' region of the core sequence. Contains duplicates (matched parts are marked *). This region at 1.720 repeats is 3 Shows an excellent match with the repeating unit of 3.15, but a less perfect match with 33.6 shows. This means that the 1.720 satellite DNA is detected by probe 33.15. However, the reason why it is not detected by 33.6 (Fig. 20) will be explained. ( 1,720 satellite repeat units (myoglobin λ33 minisatellite repeat units) ) is too large on each side of the core (H+J>15 in equation (1)). It contains too much microtide and does not function as a multilocus probe according to the present invention].

To detect bovine minisatellites hybridizing to probe 33.15. Digest the DNA with human'ul or -pdel to extract the 1.720 satellite. It was reduced to a 46 base pair monomer that was removed by electrophoresis from the bottom of the gel. 21st The diagram shows these controls from bovine DNA! ! ! It is clear for each of the enzymes This shows that 6 A fingerprints were actually obtained (M3). However, these bands almost all lost the Aju I and Dde I sites in each repeat (possibly due to However, due to mutation of one or the other of the underlined bases, overlap ~1! ! I and -pdeI sites), the normal 1.720 DN derived from a mutant block of 1,720 satellite DNA embedded within A. It is something that This would explain the following facts: In fact, identical bovine DNA fingerprints were obtained using 31 N1 and Nakadori Q1 samples.

b. Very large fragments - 5. , making the hybrid more powerful than the smaller one. (as they contain correspondingly many 1.720 satellite repeat units) be. Extremely large bovine DNA! l! J'r piece contains 440 of these units ). In humans, band intensity does not increase continuously with size (21st figure).

C. Almost all of the bovine DNA fingerprint fragments are cut once within 1.720 repeat units. One to do! (removed by h-a I (see above, data not shown).

d, father cow (N (12) IL human = 1-μm hybridization fragments were collected in the digestate) I don't have much. This, myself, contains these mutant repeat books: 1. '? ? 0 satella i) DNA region 1, suggesting that it is deleted in the rL:Dl substance. Ru.

e. The DNA fingerprints of the company (Arashi 1) and the calf (Na 5) are almost identical. Ru. The cow is probably heterozygous for the deletion t, similar to that seen in the sire; The non-deleted chromosome (and therefore the entire band) had been accidentally transmitted to the calf. subordinate So, all these bands are i’! ! Chain IJ, similar to what occurs in humans are not independently transmitted to descendants.

Nevertheless, the five cows tested had different "satellite" DNA fingerprints. Although these unusual types of fingerprints are useful for individual identification, they are It cannot be used to obtain label information.

In equation (1), some probes are valid even if n is not necessarily equal to 3. can function. In such probes, the core-free DN At least two more repeats of at least one pair of (J, core, K) by the A sequence can be separated from the repetition of Therefore, separated by “non-core” DNA sequences A sufficiently long probe with (J, core, K) arrays arranged in the pairs 1! can be achieved.

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Roe≧” II i #y- Me IN’i j number’45 dollar Emi, l l II- My eIlIl =N = B Eye No. 4 1 Kasu ”:”　H1#pls4@ ≧−t It l # glance [ Do 1 Shi 1 Ku≧〜゛゛　1　・ =-■! Rumor 11/11 Mockery 5'CTGGTGGGCTGGTGGG3' 3'CACCCGACCACCCGAC5'□Prope F[G, 12 F　5SDDDDFDDSSSS FIG. 13 ~ I 11 ・◆ - arrogance A 6 6 Akira し（〜　− F [G, 1/.

20- 41m14・ FIG. 15 123 M S S 7 8 glo 11FIG, 15△ 123 ξ 5 ===; , $−=2=・ FI[3,17 123[23 χ33・(,x　33−1r FIG. 18 ・　・−１−− x 33・ra x 33・Is’ FIG, 19” (8 ζ unchanged) x 3EL (3w 331S” y 3 3 et al x3?3/fFfG, 20 ― X　χ　×　／　X　× Procedural amendment (formality) %formula% 3. Person who makes corrections Relationship to the incident: Patent applicant Name: Lister Institute of Preventive Medicine 4. Agent Address: No. 5, Toranomon-chome 8-chome, Minato-ku, Tokyo 105, Date of amendment order February 24, 1986 (Shipping date) 6. Subject of correction (1) A representative of the patent applicant in writing pursuant to the provisions of Article 184-5, Paragraph 1 of the Patent Act. ” column (2) Power of attorney (3) Translation of the specification (4) Translation of the scope of claims (5) Corporate certificate (6) Translation of drawings 7. Contents of correction (11(2)(5) As per attached sheet) (3) Translation of the specification (no change in content) (4) Translation of the scope of claims (no change in content) (6) Engraving of translated text of drawing (no change in content) 8, attached document inventory (1) One copy of the corrected document pursuant to the provisions of Article 184-5, Paragraph 1 of the Patent Act (2) Power of attorney and its translation: one copy each (3) One translation of the specification (4) One translation of the scope of claims (5) Corporate certificate and its translation: 1 copy each (6) Translation of the engraving drawing: 1 copy international search report -h11111^n a++an'=PCT/GB 851004? )

Claims (1)

[Claims] 1. The following general formula read in the sense of 5'→3': H. (J.Core.K)n.L( 1) [During the ceremony, "Core" refers to a sequence having 6 or more contiguous nucleotides; The following sequences are read with the same meaning: [There is an arrangement] (2) [There is an array] (3) [There is an array] (4) [There is an array] (5A) [There is an array] (5B) (wherein, X is A or G, Y is C or T, T is T or U, and m is 0.1 or 2, p is 0 or 1, q is 0 or 1, and n is 3 or more. selected from one of the following; J and K together represent 0 to 15 additional nucleotides within the repeat unit. And H and L each represent zero or more additional nucleotides located at the ends of the repeat sequence. and the following, namely: (i) "Core" and J and K are the same sequence in each (J.Core.K) repeating unit or is not necessarily required to have a length; (ii) "core" can also refer to a variant core arrangement; (iii) The actual total core sequence in all n repeats is divided into the same number n repeat units. 70% or more of the “true” total core array defined above for Equations 2-5. Polynucleotides having homology] and sequences complementary thereto polynucleotide. 2. The total number of nucleotides present in each (J.Core.K) repeat sequence is 25. A polynucleotide according to claim 1, not exceeding the scope of claim 1. 3. Polymer according to claim 1, wherein the core has up to 16 nucleotides. nucleotide. 4. Claim 1, wherein the core represents a sequence of 7 or more consecutive nucleotides. The polynucleotide described in Section. 5. Claims in which the core represents a sequence of 12 or more said consecutive nucleotides The polynucleotide according to scope 1. 6. 1 in which the core is selected from the sequences shown in formula (2), (3) or (4); Claim 1 representing a sequence of 4 to 16 consecutive nucleotides. polynucleotide. 7. The actual core sequence has 80% or more homology with the true core sequence, A polynucleotide according to any claim. 8. According to claim 7, J is 0 or 1 and K is 0 or 1. polynucleotide. 9. The total number of nucleotides present in each (J.Core.K) repeat unit is 20. A polynucleotide according to any preceding claim, not exceeding. 10. The following general formula read in the meaning of 5'→3': H. (J.Core.K)n.L (1) [During the ceremony, “Core” represents a sequence of 6 to 16 contiguous nucleotides that are read interchangeably. I: (1) Human or animal genomic DNA of about 33n per repeat unit. By probing with a probe containing the myoglobin tandem repeats of t. a common core region of a first human or animal minisatellite obtained by (2) Human or animal DNA containing a tandem repeat sequence comprising (1) a second human or animal minisatellite obtained by probing with a probe Common core area of ITE; (3) Human or animal genomic DNA, a tandem repeat sequence comprising (2) A third human or animal miniature obtained by probing with probes containing Satellite common core area; selected from the tandem repeat sequence consists of three or more units] and a polynucleotide having a sequence complementary thereto. 11. The following general formula read in the meaning of 5'→3': H. (J.Core.K)・L( 1) [During the ceremony, “Core” is a collection of human or animal genomic DNA that has a consensus of 75% or more. 6 or more consecutive nucleotides from within the common core region of several minisatellites Indicates any of the sequences having; The “core” need not necessarily have the same sequence in each repeating unit; hand All other symbols are as defined in claim 1.] a polynucleotide having a sequence complementary to this; Do. 12. The following formula: (5') [There is an array] (3') (6) (In the formula, P is not G, and W is A or T or U and X is A or G) 6-36 nt containing a contiguous (5'→3') core sequence selected from Polynucleotides having three or more repeats of the sequence, or variants thereof (individuals in all repeats) The total core sequence of quality is the “true” one defined for equation (6) within the same number of iterations. (provided that it has 70% or more homology with the total core sequence); A polynucleotide whose sequence is homologous to that. 13. Claim 12, wherein each iteration or variant iteration comprises the arrangement of formula (6). polynucleotides. 14. Consecutive (5'→3') array: [There is an array] (7) is conserved in all repeat units, and P and W are defined in claim 12. polynucleonucleotides according to any of the preceding claims, having the meaning given to them. Chido. 15. Consecutive (5'→3') array: [There is an array] (8) is conserved in all repeat units, and T is T or U. A polynucleotide according to any of the claims. 16. According to any one of claims 12 to 15, wherein P is T or U. polynucleotide. 17. The porine according to any one of claims 12 to 16, wherein W is A. Creotide. 18. Recited consecutive core sequences or conserved sequences are identical to repeated sequences The polynucleotide according to any one of claims 12 to 17. 19. Next consecutive 5'→3' core array: [There is an array] (7) (where P is not G, W is A or T or U, and X is A or G ) polynucleotides having three or more repeats including The “true” sum defined for equation (7) during the same number of iterations with the same total core array (provided that it has 70% or more homology with the core sequence), and A polynucleotide with a complementary sequence to. 20. W is A at the 5' end and T or U at the 3' end , the polynucleotide according to claim 19. 21. Polymorphic minisatellites - polynucleotides with length-specific binding properties A manufacturing method comprising: (i) hybridization restricted to other polymorphic DNA regions; identifying natural tandem repeat sequences in DNA that can be fused; (ii) a natural consensus core sequence of repetitive sequences hypothetically responsible for such binding; identify, and (iii) lower genome-locus specificity and higher polymorphic fragment susceptibility than natural repeat sequences; Derived from a natural consensus core sequence with minisatellite binding that exhibits tolerance isolating or artificially creating complete or incomplete tandem repeat sequences that A method comprising: 22. A polynucleotide defined in any one of claims 1 to 20 22. The method of claim 21, wherein the method is as follows. 23. A useful point in determining the genetic origin of samples containing human or animal DNA. nucleotide probes containing a labeled or marker moiety and , corresponding fragments obtained by fragmentation of sample DNA with restriction endonucleases. be sufficiently human to allow hybridization of the probe to the DNA fragment. 3 or more tandem sequences homologous to minisatellite regions of human or animal genomes. a polynucleotide (including variants) comprising: About: a) Each of said repeats is present in a number of minisatellites from different genomic loci. A core that is 70% or more homologous to an existing consensus core region of similar length. b) said core is 6 to 16 nucleotides in length; c) contributes to the core; the total number of nucleotides within the repeat unit is 15 or less; A probe characterized by: 24. Arrangement in which the core is continuous from 5' to 3': [There is an arrangement] (7) Or [There is an array] (8) (wherein P is not G and W is A or T or U) Probe according to paragraph 23. 25. The polynucleotide is a polynucleotide according to claims 1 to 20. claim 23, wherein at least the repeating unit is of single-chain type. Probes described in Section. 26. A labeled product as defined in any of claims 1 to 20. Polynucleotides consisting of polynucleotides in which at least the repeating unit is single-stranded. Tidoprobe. 27. According to any one of claims 23 to 26, which is in the form of a single chain as a whole. probe. 28. Useful in determining the genetic origin of samples containing human or animal DNA A method for manufacturing a probe according to any one of claims 1 to 20, or the polynucleotide produced by the method of claim 21 is labeled or A method comprising introducing a marker. 29. A method for identifying a sample of human or animal genomic DNA, the method comprising: A is searched for by the probe according to any one of claims 23 to 27, and detecting hybridized fragments of said DNA. 30. The detected fragment damages the tandem repeat sequence of the sample DNA. Claim No. The method described in Section 29. 31. Two or more types of probes according to any one of claims 23 to 27 Comparing patterns of positive fragments obtained using The method according to item 30. 32. A minisample of the human or animal genome associated with an inherited disease, abnormality, or trait. Locus specific to the tellite region and of claim in family or pedigree analysis Individual patterns formed by one or more probes in the range 23 to 29 through the isolation of bands associated with the disease, abnormality or trait observed by comparison of probe obtained by 33. One or more probes according to any one of claims 23 to 27 and associated with chromosomal or DNA abnormalities associated with cancer. Probes derived from observed fingerprint bands. 34. The probe according to any one of claims 23 to 27 or claim 33 A variant of (J.Core.K) in which at least one pair of tandem arrays of (J.Core.K) is a core. separated from at least two other sequences by an array that does not contain Hence the transformation where n is not necessarily equal to 3 and all other constraints exist.