GB2225112A - Hybridisation probes - Google Patents

Description

- 1 M RIDISATION PROBES 2'Z_ 2 5 112 The present invention relates to a

method for detecting the presence or absence of at least one variant sequence in a single test and to kits for use in such a method.

The present invention is of particular interest in the diagnostic screening of DNA samples for inherited conditions.

Several hundred genetl'c diseases are known to exist in man which result from particular mutations at the DNA level. The molecular basis for certain of these diseases is already known and research is rapidly revealing the molecular basis for those genetic diseases for which the nature of the mutation is at present unknown. Where the precise molecular basis for the inherited condition is not known, diagnosis of the disorder or location of carriers may be provided by RFLP technology. Thus, at present Duchenne Muscular Dystrophy, Cystic Fibrosis and Huntington's Chorea deficiency may for example be diagnosed using RFLP technology. Such testing however needs to be performed separately in respect of each condition and a substantial amount of work is required, each case requiring inter alia Southern blotting, hybridisation and pedigree analysis. Certain other innerited conditions are known-to be associated with single point mutations in genes, but each of these conditions must be analysed separately and particular difficulties arise where the point mutations are heterogeneous. Thus for example more than 20 different point mutations can cause beta-thalassaemia and at least 5, and probably many more than 12, point mutations can cause haemophili A.

The present invention is based on the discovery of a method which ameliorates the above-mentioned difficulties by enabling the presence or absence of at least one variant sequence to be detected in a single test and thus may be of interest in the diagnostic screelting of DNA samples for inherited conditions including predispositions.

Thus according to one feature of the present invention there is provided a method for detecting the presence or absence of at least one variant sequence in a single test, which metho, comprises: 1) hybridising a detectable first nucleotide probe and, a secon,' nucleotide probe to adjacent segments of each desired diagnostic portion of a target base sequence, tlie iiLic:lcc,,ide seqtiei-,2e of tilic firs, and second probe being such that where a hybrid of the first a= Secone- z 2 - probes is formed with a diagnostic portion of a target base sequence the first and second probes are separated by a gap, which gap is defined by at least two nucleotides of the target sequence which nucleotides of the normal and/or variant target sequence comprise all but one of the different naturally occurring nucleotides or less; 2) contacting the diagnostic portion of the target base sequence with one or more nucleotide species under conditions enabling complementary hybridisation, the one or more nucleotide species used being such that either the normal or the variant nucleotide is non-complementary therewith; 3) subjecting any hybrid obtained to linkage; and 4) determining the presence or absence of each variant sequence by detecting the presence or absence of probe(s) characteristic of the presence or absence of each variant sequence; the method being effected such that the nucleotides defining the gap between the first and second probes hybridised to the said diagnostic portion of the target base sequence are selected to permit linkage of the first and second probes in respect of either the normal or the variant target sequence but not both such sequences.

A hybrid of the first and second probes hybridised to the target sequence with.a gap between the probes as defined above is referred to herein as a "split probe hybrid" and similarly the first and second probes in their unhybridised state are referred to herein as "split probes".

The expression "variant sequence" is used herein to include a single nucleotide as well as a plurality of nueleotides. In relation to the detection of inherited conditions the expression will generally mean one or more nucleotides which are associated with the presence of or heterozygosity for an inherited disorder or predisposition. It will be appreciated that the detection of the presence of a variant sequence may be achieved either by the positive detection of the said sequence by detecting the linkage of the appropriate split probes or by detecting the absence of a known normal sequence b., detecting the failure of the appropriate split probes to link. Similarly the absence of a variant sequence way be detected either by detecting the presence of a known normal sequence by detectiri, the linkage o t.-,c 11 appropriate split probes or by detecting the absence of the variant sequence by detecting the failure of the appropriate split probes to link.

Thus for example the presence or absence of the desired variant sequence may be determined by detecting any linked probes having a sequence which is homologous, preferably completely homologous, with the portion of the target sequence to which they are hybridised, such linked probe characterising the diagnostic portion of the target sequence to which they are hybridised.

In respect of each potential variant sequence the first nueleotide probe must be detectable, but the second nucleotide probe may or may not be detectable as desired.

The term Metectable" is used herein to mean capable of detection. Thus the first nucleotide probe need not carry a signalling means such as a radioactive label or a non-radioactive signalling complex, although such may be present, provided that the probe may subsequently be treated to render it capable of signalling.

The expression "diagnostic portion" as used herein means that portion of the target base sequence (as hereinafter defined) which contains the potential variant sequence, the presence or absence of which is to be detected.

The expression "target base sequence" as used herein means a nueleotide sequence comprising at least one diagnostic- portion (as hereinbefore defined). Thus for example in a single test for beta-thalassaemia the target sequence may contain up to 50 diagnostic portions, each diagnostic portion containing a single potential -;ariant sequence. Where a target base sequence is present which contains only a single potential variant sequence, the method will be effected using more than one target base sequence.

One feature of the present invention is that the probes for detection, obtained according to the method oil the present invention, characterise the diagnostic portion(s) of the target sequence to which they are hybridised. Thus for example the linked probe hybridised to one diagnostic portion of the target sequence is distinguishable fror the linked probes hybridised to other diagnostic portions of the target, sequence. This may be effected by any con-.,enient means. ThuS ic,r i i ere i.

example each detectable first nucleotice probe ma.. carr.v a d.- and distinguishable signal or capable of a Such signals and residues capable of producing a signal are discussed in detail hereinafter, but might for example include the solid phase amplification system described by Wang C G in World Biotech Report 1986 vol. 2, part 2, pages 33-37, (Diagnostics Healthcare Proceedings of the conference held in November 1986, San Francisco) in which microbeads formed with many chosen trace elements are conjugated to the probe. The presence of specific linked probes may be detected by x-ray fluorescent analysis. A much simpler and preferred method of rendering the probes, for example the linked probes distinguishable is however to ensure that the length of each detectable probe, e.g. the length of each detectable linked probe which may be formed during the method of the present invention, is different. The presence or absence of a given potential variant sequence may thus advantageously be detected by electrophoretic techniques, in which the different detectable probes, e.g. the different detectable linked probes obtained may be distributed according to their molecular weight and thereby identified for example by autoradiography. The lengths of the linked probes may only differ by a single nueleotide, but preferably the lengths will differ by at least 3 nucleotides.

The detectable fLrst nu--'LeOtide probe and the second nucleotide probe are-such that they hybridise to adjacent segments of each desired diagnostic portion. It will be appreciated in this regard that within each diagnostic portion the potential -,-ariant sequence is present between that segment to which the detectable first nucleotide probe hybridises and the segment to which the second nucleotide probe hybridises.

It will be appreciated that whilst the first and second probes need not be completely homologous to the diagnostic portion to which they are intended to hybridise provided that they are in fact capable of hybridisation. complete homology is preferable. In any event it is preferable that the nucleotides of the first and second probes adjacent the gap be completely homologous to the diagnostic portion to which they are intended to hybridise.

The gap between the probes is defined by at least two nucleotides of the target sequence, which nucleotin_es of the normal and/or variant sequence comprise all but one of the different natura-'-"..' occurring nucleotides or less. Moreover. the one- or more nucleotide species employed to contact the diagnost-ic portion of the tarEet bap- 1 1 sequence in the method of the present invention are such that either the normal or the variant nucleotide(s) is non- complementary therewith. These requirements characterise the size of the gap between the probes and set the minimum gap as two nucleotides, the maximum gap being dependent on the nature of the nucleotides of the target sequence which define the gap.

Furthermore the position of the normal or variant nucleotide within the gap is selected to avoid or at least reduce the chance of linkage across a gap of a single nucleotide without filling in of the gap by the relevant complementary nuclotide. This situation may arise where the first and second probes are hybridised to the diagnostic portion of the target base sequence to define a gap of at least two nucleotides as hereinbefore defined and the method of the present invention is effected to fill in the gap such that a gap of only a single nucleotide, is last formed in which the sole nucleotide defining the gap is either the normal or the variant nucleotide to be detected.

It is then possible that linkage of the first and second probes may take place regardless of whether the 1 nueleotide gap has been filled or not with the result that it is not possible to reliably distinguish between the presence of a normal and a variant nucleotide. It is thus important in the prejent invention to so position the normal or variant nucleotide within the gap that if the method is effected in the presence of an agent for polymerisation which initiates syntnesis in the direction 5' - 3' a nucleotide corresponding to the normal or variant nucleotide to be detected is not introduced into the terminal 3' position of the gap. If this is not ensured then the first probe will be extended by the appropriate number of nucleotide(s) to fill in the gap except for the 31 terminal position, thus leaving a single nucleotide gap with the result that there is a ris-- of linkinE the first and second probes across the gap regardless of whether the gap has been filled in or not. Similarly if the gap is defined b.-,- two or more nueleotides and the method is effected in the presence of an a.en for polymerisation which unusually initiates synthesis in the direction 3' - 5' a nucleotide corresponding to the norr,-,'- --- variant nucle- tide to be detected shou'id not be introduce inte the 'er!7nal- 3' p-sition of the gap. The nucleotides of the target. sequence which define the gap between the split hybridised to the target --eqii-2n-C are effective to per!nit of the probes- in respect -:- ti-,j normal or the variant target sequence but not both such target sequences, whereby the presence or absence of a variant sequence may be detected.

Thus, for example the gap may be of two or three nucleotides, which may be the same or different, or where one or more of the two or three nucleotides is repeated the gap may be as large as desired consistent with the above-mentioned characteristics. By way of example the gap between the split probes may be defined by the sequence TC in the normal target sequence and by GC in the variant target sequence. Contacting the diagnostic portion of the normal target base sequence with the individual nucleotides G and A under conditions enabling complementary hybridisation, followed by linkage of the first and second probes will result in formation of a single probe of a leng-th equivalent to the lengths of the first and second probes together and including the two nueleotides which have filled in the gap between the first and second probes. Contacting the diagnostic portion of the variant target base sequence with the same individual nucleotides G and A under conditions enabling complementary hybridisation followed by an attempt to link the first and second probes will not result in the length of the first probe being extended thus no linkage of the two probes will take plaCe. It will thus be possible to distinguish the normal target base sequence from the variant base sequence by detecting the presence of a single probe of the calculated length in respect of the normal sequence and by detecting the presence of a single probe of the shorter calculated length or if desired two separate short length probes in respect of the variant sequence. A similar example may include a gap between split probes de fined by the sequence ACTA in the normal target sequence and by ACGA in the variant target sequence, the individual nucleotides T, G and A being used to contact the diagnostic portion of the target base sequence. Similarly for example the gap between the split probes way be defined by the sequence AWT in the normal target sequence and by ACC-L in the variant target sequence, the individual nucleotides T, G and A being used to contact the diagnostic portion of the target base sequence.

Nucleotide(s) complementary to the targe,, sequence between adjacent segments to which the first and second nucleotide probe-s are Prezence of h,.,br-i-dise,-' ra.: be polymerase. for example tt-lc- frag,-,.c-it of D!.t^. 1 o.r calf- thymus DNA polymerase alpha but preferably Taq polymerase. The first and second nucleotide probes may be linked by any convenient means known per se such as for example by enzymatic ligation using, for example, a DNA ligase or by covalent/non-covalent linking using for example a biotin- avidin cross-link, but are preferably linked by ligation for example with DNA ligase.

Nucleotide(s) complementary to the target sequence between adjacent segments to which the first and second nucleotide probes are hybridised are introduced under hybridisation conditions such that non-complementary nucleotide are not incorporated. The method of the present invention can be effected using only a single nucleotide e.g. to hybridise to an AA, GG, CC or TT gap but is generally effected in the presence of two different nueleotides. Conveniently the method would be effected using a separate reaction vessel in respect of each pair of nucleotides to be introduced. Thus up to ten reaction vessels may be employed, one for each pair of nucleotides. Since it would be known which specific pair of nucleotides had been introduced to form a linked probe, it will be possible to determine whether a normal or a variant sequence was present at the relevant diagnostic portion of the target sequence. It, such a case the DNA polymerase used will ad,:antageously be the Klenow fragment of DIZA Polymerase I or calf thymus D,,A. polymerase alpha, preferably Taq polymerase. Conveniently the MA polymerase will ha,,e no significant eXonuclease acti%,it-,;.

While the present invention is most useful for directing oligonueleotides to specific hybridisation sites in complex genomes in order for example, to analyse for the presence of point mutations it is also of use for analysis of other variations in compley genor-..e-c such as translocations and such analyses may be effected together with the analysis of for example point mutations in the same test. For t the non-detectable second pol-;nueleotide of a split probe will 1-,a-,-e one n.eri,ber of a binding pair associateG therewith and;ill hybridise to genomi, sequences adjacent to potential translocation breakpoints. The detectable probe will preferably b- up te long and span a region of potential translocation breakpoints. Quant-Ltat-i-.c- - - ---- J -,'- - - - linkag. PrebC hybridisation with nucleotides complementary t- the gap. of a., leaSt.

analysis of translocations, it is preferred tha c 11 nucleotides between the adjacent ends of the first and second nucleotide probes will only occur where the genomic region is contiguous and uninterrupted by translocations. The occurrence of a translocation will preclude a proportion of the detectable probe from linking to the non- detectable polynueleotide. On subsequent denaturation of hybridised polynucleotide and application of the other member of the binding pair, a proportion of the detectable polynucleotide will not then be found to be associated with the polynucleotide complexed with the other member of the binding pair. The binding pair will preferably comprise n antigen- antibody or a biotinavidin interaction whereby, for example, denatured hybrids are passed through a solid phase complexed with one member of the binding pair and the eluate analysed for the presence of the detectable probe.

Thus in a further embodiment of the invention we provide a method which includes at least one analysis of a translocation in a target base sequence e.g. a complex genome in which the detectable first nucleotide probe is adapted to hybridise across a region of potential translocation(s) and the non-detectable second nucleotide probe is adapted to hybridise to sequences adjacent to said potential translocation(s), the non-detectable second nucleotide probe carrying one member of a bindIng pair, the method comprising formation of a split probe hybrid which is subjected to hybridisation with nucleotides complementary to the gap of at least two nueleotides between the adjacent ends of the first and second nucleotide probes and then linking to form a linked probe hybrid.

If desired each of the detectable first nucleotide probe and second nucleotide probe may carry a moiety such that after linked probe hybrid formation, and denaturation where appropriate, a signal is only detectable if a nucleotide sequence is obtained which carries both the moiety attached to the detectable first nucleotide probe and the moiety attached to the second nueleotide probe. The nucleotide probes may for example be oligonucleotide probes.

This technique might for example be effected using nonradioactive energy transfer procedures (see for example European Patent Publication No. 70685 of Standard Oil Co.) or enz-;me channellintechniques (see for example European, Patent Publication No. 95A7 of Syva Co.).

The method of the present invention may also include an analysis in which a target sequence or diagnostic portion thereof is subjected to hybridisation with more than two oligonucleotide probes followed by hybridisation with nucleotides complementary to the gap(s) of at least two nucleotides between adjacent ends of the oligonucleotide probes such that where a complementary target sequence is present a split probe hybrid is obtained, the detectable first nucleotide probe being a terminal oligonucleotide probe which is detectable and the second nucleotide probe being the other terminal oligonucleotide probe which has attached thereto one member of a binding pair, individual other oligonucleotide probes(s) being hybridised separately but adjacently to a contiguous target sequence between the said terminal oligonucleotide probes, and the individual oligonucleotide probes linked to join the detectable oligonucleotide probe to the oligonucleotide probe having one member of a binding pair attached thereto to form a linked probe hybrid which is then denatured and contacted with the other member of the binding pair whereby the detectable linked probe nucleotide sequence including the said binding pair may be separated from other nucleotide sequences. Thus one terminal oligonucleotide (hybridising adjacent to a series of other oligonucleotides) f6r example has attached thereto a detectable signalling moiety while the other terminal oligonucleotide has attached a moiety forming part of a binding pair the moiety being eftective to enable the oligonucleotide to be recovered in solution from a mixture containing other nucleotide sequences. Adjacently hybridised oligonucleotides are linked together by appropriate treatment in order to effectively join the signalling and binding moieties via a linked probe. Loss of any individual oligonueleotide hybrid results in subsequent failure to join the signalling and binding moieties. The linked probe hybrid is denatured and contacted with the other member of the binding pair which recognises one terminal oligonucleotide in the linked probe. The linked probe may thus if desired be separated from other oligonueleotides in the mixture and then analysed for the presence of the detectable moiety.

Thus the association of the binding oligonucleotide with thi- signalling oligonucleotide is dependent on the presence. upon linking 0.6 f adjacently hybridised oligonucleotides. of all adja--cnt oligonucleo tides between the hybridised signaIling oligon,,icle-e.5c a-., C_ the binding oligonucleotide. The binding pair effecting separation of the linked probe with an associated binding moiety may be avidin and biotin or an antigen and associated specific antibody. This embodiment provides for analysis of longer stretches of target nucleotide sequence than with just two adjacent hybridising linked or split probes as in other embodiments.

Where it is desired that the nucleotide probe should carry a signal or a residue capable of producing a signal, such signals or residues may be known per se e.g. a radioactive label.

The residue which is capable t of producing a nonradioisotopic signal may alternatively comprise a signalling part normally separated from the oligonucleotide by a spacer group. This spacer group may if desired be linked to the signalling part of the complex by a direct covalent link or by a protein-ligand or antigenantibody interaction, for example, an avidin-biotin or dinitrophenylantidinitrophenyl antibody interaction. It will be understood that the signalling part of the residue may itself be capable of signalling or may be capable of producing a signal by interaction with an appropriate agent according to methods known per se. Thus, for example a preferred signalling part of the covalently attached residue incorporates a system for producing'an enzymatically-activated production of colour change. Preferred enzyme systems involve alkaline phosphatase, acid phosphatase, beta galactosidase, luciferase, or horseradish peroxidase. Such enzyme systems are not themselves capable of signalling, but are capable of producing a signal in the presence of an appropriate substrate according to methods known per se. The signalling part of the covalently-attached residue may operate according to any conventional technique such as for example by luminescence, fluorescence or by means of colour. Where the enzyme alkaline phosphatase is employed a particularly convenient substrate is the chemiluminescent substrate system described in European Patent Application, publication no. 254051 (Wayne State University) and by Schaap et al, Tetrahedron Letters, 28, 11, 1155-1158.

In use it will generally be necessary for the nucleic acid probe to detect minute amounts of the fully complementar.'. oligonucleotide sequence. In such circumstances it,.-ill be advantageous to incorporate within the probe a means of amr-lifyin,7 the signal. The amplification can bt carried out by known technique--, for c example using one or more of the systems described in European Patent Publications 27036 (Self), 49606 (Self), 58539 (Self) and 60123 (Self).

The presence of any linked probes may be detected by any convenient method, but will be dependent on the signalling means or residue capable of producing a signal carried by the detectable probes. For example the linked probes may be separated from the target nucleotide sequence to which they are hybridised by denaturation, if necessary by selective denaturation and the linked probes analysed by for electrophoresis and where appropriate autoradiography. Detection may also be effected by the use of a binding pair. Thus for example the first or second nucleotide probe may if desired have one member of a binding pair. Generally the one member of a binding pair will be carried by, or be part of, the second nucleotide probe. The other member of the binding pair may be in solution or on a support. Thus where appropriate, the second nucleotide probe linked to the detectable first nucleotide probe may be isolated on a support carrying the other member of the binding pair. Such a binding pair may for example be a protein-ligand or antigen-antibody interaction such as an avidin-biotin or dinitrophenyl-antidinitrophenyl antibody interaction.

indeed one member of the bi-ding p----r may be a nuclectide sequence, which sequence may be a portion of the probe sequence or rra- be a nucleotide sequence branch on the probe. The ether member of the binding pair may for example be a protein which binds to the nucleotide sequence or another nucleotide sequence to which it hybridises.

Thus the method of the present in-.-enior. enaLles many individuals to be analysed for inherited conditions including heterozygote detection for example if both a suspected "normal" oligonueleotide to be detected and a suspected "mutanC oligonucleotide to be detected are substituted at the appropriate site. Moreo-,-c,. the preferred method of the invention enables the procedure to be completec. substantially more rapidly than standard RFLF analysis since the need for Southern blotting is obviated and filter hybridisation may be avoided.

Inus for example where a gene imay contain a gi,.yen number X known point mutations, any one of which cause-s a particula-- inheritel condition the P,e-Li-iod of D-- tC, g-".'E:

- Thus X oligoiiuc-lec,tirJj-j,-. e.g. 31-, a diagnosiZ.

I_ synthesised each complementary for example to the 3' side of the possible mutation points. Each oligonucleotide will therefore have a different sequence. Each oligonueleotide may be labelled, for example 51-labelled, conveniently radioactively labelled (e.g. with 32 P). X further oligonucleotides may then be synthesised, each of different lengths for example having lengths 5 nucleotides apart e.g 15, 20, 25 and 30 nucleotides and each having a terminal residue adjacent to the suspected mutant and its corresponding labelled probe, for example a 5, terminal residue base. This second set of oligonucleotides will also be capable of hybridising with th normal DNA sequence. The oligonucleotides will be designed to hybridise to either side of the suspected point mutation such that a split probe hybrid is formed with a gap of at least two nueleotides between the probe portions of the hybrid. Total human D1,A may then be taken, denatured and all 2X oligonueleotides added to a single reaction vessel. The oligonucleotides may then be hybridised to the denatured DNA and one or more nucleotide species added under conditions enabling complementary hybridisation followed by ligation. The ligation mixture may then be denatured and subjected to electrophoresis e. g. loaded onto a denaturing polyacrylamide gel. Autoradiography may then be effected so that the presence or'absence of possible point mutations may be visualised. In respect of normal WA no ligation will take place where the nucleotide species added are complementary to the corresponding mutant sequence, and thus the longest sequences visualised will be no longer thanthe longest oligonucleotide starting materials. Only the presence of a point mutation will result in ligation and thus the presence of a longer nucleotide sequence. If however the second nucleotide probe is labelled, this probe may be extended to a limited extent but appropriate choice of the primer length in the context of the diagnostic region will obviate longer extension products that may be mistaken for ligation products. The size of the product band (i.e. the length of the nucleotide sequence) will characterise absolutely the nature of the mutation.

It will be appreciated that the first set of Y oligonueleotides hereinbefore described could equally well be designe-such their 31 terminal residues are adjacent to the suspected mutant base. Selective ligation to a second set of a further X C oligonucleotides of increasing chain length as above might then be effected and detected as described.

It will be also be appreciated that either of the two sets of X oligonueleotides may be synthesised having for example lengths 5 nucleotides apart. In general the set of oligonucleotides of constant length will be labelled as hereinbefore described.

Confirmation of the presence or absence of a given point mutation may be obtained by repeating the above procedures but substituting an appropriate set of nucleotides which are fully complementary to be expected normal seqpence. A combination of the two approaches provides a method for the detection of heterozygotes which will be of value for the analysis of dominant inherited conditions and in the detection of carriers of recessive inherited conditions.

Examples of base-pair substitutions causing genetic disease which may be detected using the method of the present invention include the following:

TABLE Base-pair substitutions causing genetic disorder A Autosomal Disorder Base-pair substitution Antithrombin III deficiency CG - TG t Amyloidotic polyneuropathy CG -) CA AC --> GC AG - GG Alpha-1-antitrypsin deficiency TC - CG CG -> CA Adenosine deaminase deficiency CG -> CA AA -> AC Reference Duchange et al j Nucleic Acids Research 14:2408 (1986) Media S. et al., Mol.Biol.Med, 329-338 (1986) Wallace M.R. et al, J.Clin.Invest. 78:6-12 (1986) Wallace M.R. et al, J.Clin.Invest 7S:6-12 (1986) Nukiwa T. et a!., J.Biol.Chem. 261: 15989-15994 (1986) Kidd V.J. et al., Nature 304:230-234 (1983) Bonthron D.T. et a!.. J.Clin Invest 76:894-897 (19C_) Valerio D. et al EM--,-, 5:113119 (198f-1) Disorder Apolipoprotein E deficiency Diabetes mellitus, mild Gaucher's disease type 2 Hyperinsulinaemia, familial CC -> CA CA -), CA' Immunoglobulin kappa deficiency Base-pair Reference substitution CT -> CC 11 CT --> CC Cladaras et al, J.Biol.Chem 262:2310-2315 (1987) TC ->CC Haneda M. et al., Proc.NatLAcad.Sci.USA 80:6366-6370 (1983) TC---5 TC, Shoelson S. et al., 1,'ature 302:54C,-543 (1983) TG -y TT S. et C-1 > GC 'is Uj L lZ.Engi.J.Med 1, al., Sliiba-.:aki Y. et al.,J.Clin.Invest.

Chan S.J. et a' - _L I! Proc.Nat'L.Acad.Sci. US-. 84:2194-2197 (19E,') CT -> CC Sta-,;nc-z:e,:-1ordgren J. et al., 458-4,',! (198) CG, > ', Science 230:

c Disorder Base-pair Reference substitution LDL receptor deficiency GG GA Lehrman M.A. et al., Cell 41:735-743 (198-5) Osteogenesis imperfecta TG TT Cohn D.H. et al., (type II) Proc Natl Acad Sci USA 83: 6045-6047 (1986) Phenylketonuria AG AA Dilella A.G. et al Nature 3221:799-803 (1986) CG TG Protein C deficiency Purine nucleoside phosphorylase deficiency Sickle cell anaemia Dilella A.G. et al., Nature 327:333-336 (1987) C- r - -> 1 1 GC. - GC Romeo G. et a! Prcc Natl Acad Sci US...'. 84., 2829-2832 (1987) f' TC --> TA Williams S.E. et a!., J.Biol.Cher-26 2332-2339 (1987) GAG -) GTG Chang J.C. and Kan Y.; Lancet 2, 1127-9 (1981); Orkin S.H. et al., New Eng.J.Med. 307, 32-6 (1982); and Clonner E.2. e al Prc.c.

Nat.ica--Sci. USA.. 8!-.

Disorder Base-pair Reference substitution Tangier disease AC - AT Law SW. Brewer HB J.Biol.Chem 260:

12810-12814 (1985) beta-Thalassaemia Mutant Class Type Reference 1 1 a) non-functional mRNA Nonsense mutants (1) codon 17 (A - T) 0 (2) codon 39 (C - T) (3) codon 15 (C - A) ( ú4) codon 37 (G - A) 0 (5) codon 121(G - T) Chang J.C. et al Proc.,atn.Acad.Sci. USA 76: 288( (1979) Trecartin R.F. et al J.Clin.In.-Est. 68: 1012 (1981) and Cheliab F.F. et a! Lancet i: 3 (198C) Maz.azzian H.H. et a', 3: 593 Boehr... C.D. et al Blood 67: 1i8-11 (198.-) lazaz:ia.,-. F.H. et al. A-.J.Hur.Genet. 38: A860 ( 19 _-, t) Frameshift mutants (6) - 2 codon 8 Orkin S.H. et a]., j:.7 E2 (1q811 c Mutant Class Type Reference (7) - 1 codon 16 0 (8) - 1 codon 44 0 (9) - 1 codons 8/9 0 (10) - 4 codons 41/42 0 (11) - 1 codon 6 0 (12) + 1 codons 71/72 0 (b) RNA processing mutants Splice junction changes Kazazian H.H. et al Eur.Molec.Biol.Org.J. 3: 593 (1984) Kinniburgh A.J. et al Nucleic Acids Res 10: 5421 (1982) k Kazazian H.H. et al Eur.Molec.Biol.Org.

J.3: 593 (1984) and Wong C Et al Proc.Natn.Acad.Sci.

USA 83: 6529 (1986) Kazazian, HH et al Eur.Molec.Biol.Org. J 3: 593 (1984) and Kimura A. et a! J.Biol.Chem. 258: 2746 (1983).

Kazazian H.H. et aI Am.J.Hum.Genet. 35: 1028 (1983) Cheng T.C. et al Proc.Natn.,'.cad.Sci. US4 81:

28-21 (1984) 19 - Mutant Class Type Reference (1) IVS 1 position 1GT->AT 0 (2) IVS 1 position 1 GT->TT 0 (3) IVS 2 position 1 GT->AT 0 (4) IVS 1 31 end:-17 bp 0 (5) IVS 1 3' end:-25bp (6) IVS 2 31 end: AC, ->CC 0 (7) IVS 2 31 end: AG->W Orkin S.H. et al Nature 296: 627 (1982) and Treisman R. et al Nature 302: 591 (1983) Kazazian, H.H. et al Eur.Molec.Biol.Org.J. 3: 593 (1984) Orkin, S.H. et al, Nature 296, 627 (19822) and Treisman R. et al Cell 29: 903 (1982) Bunn H.F. et al, Haemoglobin: molecular genetic and clinical aspects, p- 283 (Saunders, Philadelphia 1986) Kazazian, E.H. et a!., Eur.Molec.Biol.Org.J.,3: 593 (1984) and Orkin, S.H. et al. J.Biol.Chem.258: 7249 (1983).

Padanilam B.J., et a] Am.J.Hematol. 22:259 (1986) Antonarakis S.E. et al Sri- USA. 81:

('Lclo'-) and Atve G.F.

et al Nucleic icids Res. 13:

11 Mutant Class Consensus changes (8) IVS 1 position 5 (G->C) (9) IVS 1 position(G --.,T) 0 (10) IVS 1 position 6 (T-C) Internal IVS changes (11) IVS 1 position 110 (G 7A) + (12) IVS 2 position 705(T-YG) + (13) IVS 2 position 745 (C-)G) + (14) IVS 2 position 654 (C 71) C) Type Reference Kazazian, H.H. et al Eur.Molec.Biol.Org.J. 3: 593 (1984) et al Nature 302: 591 (1983) and Treisman R k Wong C. et al Proc.Natn.Acad.Sci.USA 83:

6529 (1986) and Atweh G.F.

et al Nucleic Acids Res.13:

777 (1985).

Orkin S.H. et al Nature 296:

627 (1982) and Atweh G.F. et al Am.J.Hum.Genet. 38: 85 (1986) 1 Westaway D. et a! Nucleic Acids Res.9: 1777 (1981) Dobkin C. et a! Proc.Natn.Acad.Sci.US,t'. 80: 1184 (1983).

Orkin S.H. et al Nature 1) 496: 627 (1982) andi Treisman R. et al Nature 302: 591 (1983) Cheng, T.C. et al Proc.t.,'atn.Acad.Sc.. USA 81:

28-21 (1984) A - 21 Mutant Class (15) IVS 1 position 116 (T->G) ? Coding region substitutions (16) codon 26 (G4A) (17) codon 24 (T->A) (18) codon 27 (G4T) (c) Transcriptional mutants (1) 88 C->T (2) -87 C-)G (3) -31 A-3G Type Reference Feingold E.A. et al Ann.N.Y.Acad.Sci 445: 159 (1985) P+,PFThein S.L. et al J. Med. klene t. (in press 1986) and Orkin S.H. et al Nature 300: 768 (1982) Goldsmith et al Proc.natn.Acad.Sci USA 88: 2318 (1983) knots1 Orkin SH et al Blood 641: 3-11. (1984) + wong C. ct al Proc.Natn.Acad.Sci.USA 83: 65-20- (198E) Orkin S.H. et al, J.Biol.Cher.. '5: 8679 (1984) Orkin S.H. et al Nature 2'6: 627 (1982) and Treisman R. et al Nature 302: 591 (1983) TalAhara Y. et al Blood E7:

c I-) Mutant Class (4) -29 AG (5) -28 AC (6) -28 A-G (d) Polyadenylation mutant (1) AATAAA-AACAAA (e) Deletions (1) 310(-619bp) (2) 5'0(-1-35kb) (3) 0 (10kb) Type Reference Antonarakis S.E. et al Proc-Natn.Acad-Sci.USA 81: 1154 (1984) and Wong C. et al Proc.Natn.Acad.Sci.USA 83: 6529 (1986) t Surrey S. et al J.Biol.Chem. 260:6507 (1985) Orkin S.H. et al Nucleic Acids Res. 11: 474-7 (1983) + Orkin S.H. et al Eur.Molec.Biol.Org.J. 4: 453 (1985) 0 Spritz R.A. et al Nucleic Acids Res. 10: 8025 (1982) and Orkin S.H. et al Proc.Natn.Acad.Sc:L. USA 76: 2400 (1979) 0 HbF Padanilam B.J. et al Blood 64: 941 (1984) 0 HbF Gilman J.G. et al Br.J.Haemat. 56: 339 (1984) 23 - + = f-Thalassaemia mutant (0 which causes reduced P-globin chain production: 0 = a mutant (B') which causes absent 0--globin chain production.

Disorder Base-pair substitution Triosephosphate isomerase AG -3PAC deficiency Uroporphyrinogen decarboxylase deficiency B X-linked GG -> CA k Reference Daar 1.0. et al.,Proc.Natl. Acad.Sci USA 83:7903-7907 (1986) De Verneuil H. et al., Science 234:732-734 (1986) Haemophilia A Factor VIII CG -> TG (24) Gitschier J. et al., Nature 315:427-430 (1985) CC -3 TG (26) C, -->IG (Ib) Antonarakis S.E. et al., N.Engl.J.Med. 313: 8442 848 (1985) CC -),CL (26) Gitschier J. et al., Science 232: 1415- 1416 (1986) 1 t 11 CC --p TG (18) Youssoufian H. et al., Nature 324: 380-382 (1986) CC TC (22) 11 CC TG (22) it Haemophilia B Factor IX CC -> CA GI -> TT 1 ' 11 Eentle.; A.K. et al.. 4 Rees D.J.G. et al., Nature 316: 643-6-f-', Disorder Base-pair Reference substitution GA ->GG Davis L.M. et al., Blood 69:140-143(1987) If desired, the method of the present invention may be operated so as to amplify the signal which may be generated by the detectable probe(s). This may be achieved by effecting any linkage, for example ligation, and/or any fill in with a DNA polymerase in the presence of an excess of one or more probe set(s), each probe set comprising a detectable first nucleotide probe and a second nucleotide probe and each probe having a nucleotide sequence homologous to adjacent segments of a desired diagnostic portion of a target sequence. Thus for example the linked probe for detection may be formed, denatured, for example for detection, and further split probe hybrids formed, linked and denatured if desired ad infinitum, thereby producing the desired amplification. Therefore where, for example, the amount of genomic DNA to be tested is limiting amplification techniques may be used. Therefore DNA polymerase/ligation may be performed, for example in solution, followed by denaturation, for example heat denatur&tion, followed by the use of further DNA polymerase/ligation, the reactions being effected in the presence of an excess of the relevant probe set(s) and nucleotides.

According to a further feature of the present invention there is provided a probe set for use in the method of the present invention each probe set comprising a first detectable nucleotide probe and a second nucleotide probe hybridisable to adjacent segments of a diagnostic portion of a target base sequence, the nucleotide sequence of the first and the second probe being such that in use they form a hybrid with a complementary target sequence in which the first and second probes are separated by a gap formed by at least two nucleotides of the complementary target sequence which nucleotides of the normal and/or variant target sequence comprise all but one of the different naturally occurring nucleotides or less, the gap being further characterised in that in use one or more nucleotide species may be used to contact the diagnostic portion of the target base sequence such nucleotide species be-M6 selectable sucii that eitber the normal or the variant nucleotide(s) is non-complementary thEre--ith- The potential variant sequence contained in the diagnostic portion of a target base sequence is preferably a base-pair substitution which is, for example, indicative of a genetic disorder. In this regard probe sets based on the genetic disorders set out in the Table hereinbefore constitute a further embodiment of the present invention.

The first and second nucleotide probes are of any convenient length. The probes may therefore independently contain 5 to 50 nucleotides, for example 15-30 nucleotides. The first and second nucleotide probes are conveniently separated by up to 5 nucleotides of the target sequence. In a preferred embodiment a gap of two nucleotides is present.

In order to enable the method of the present invention to be effected conveniently it will generally be advantageous to incorporate the nucleotide probes in a kit and this kit is therefore regarded as a further feature of the invention.

Thus according to a further feature of the present invention there is provided a kit for detecting the presence or absence of at least one variant nueleotide sequence which comprises at leasi one probe set as hereinbefore defined, each probe set comprising a detectable first nueleotide probe and a second nucleotide probe, each probe having a nucleotide sequence homologous to adjacent segments of a desired diagnostic portion of a target sequence, a potential variant sequence being present therebetween the nueleotide sequence of the first and the second probe being such thal,-,,here a split probe hybrid is formed with a complementary target sequence the probes are separated by a gap formed by at least two nucleotides of the complementary target sequence.

The kit may additionally contain set(s) of one or more nucleotides which are either complementary or noncomplementary to the target nucleotide sequence bridging the first and second nucleotide probes and further containing reagent(s) for linking said probes The kit will also advantageously contain means L-r extracting D'ZA and/or means for immobilising DIZA on a solid support.

If desired at least one of the detectable first nucleotide probe and second nucleotide probe in each probe set may carry one member of a binding pair, the other member of the binding pair being present for example on a solid support provided with the kit. The kit will also comprise appropriate buffer solutions and/or washing solutions and will contain written or printed instructions for use of the kit according to the method of the present invention.

The invention may be illustrated, but not limited by the following Example and Figures wherein:- t Figure 1 illustrates the analysis of individual loci. The autoradiograph shows bands from gel electrophoresed reaction mixtures described in detail in the Example set out below. Lanes 1-6 correspond to reactions 1-6 Figure 2 illustrates the analysis of two separate loci simultaneously. Lanes 7 and 8 correspond to reactions 7 and 8. The autoradiograph shows bands from gel electrophoresed reaction mixtures described in detail in the Example set OUt below.

In both of the above Figures the sizes of the reaction products (in nucleotides) are indicated on the right. The origin of the gels is also indicated. BPB indicates the position of the bromophenol blue marker dye.

EMPLE Oligodeoxyribonucleotides (oligonucleotides) are synthesised complementary to regions within exons of the human alpha I antitrypsin ((xIAT) gene. The base pair sequence of nucleotides 1112-1329 of the gene is as follows:- T j 1112 Alul CTGCTGGGGCCATGTTTTTAGAGGCCATACCCATGTCTATCCCCCCCGAGGTC GACGACCCCGGTACAAAAATCTCCGGTATGGGTACAGATAGGGGGGGCTCCAC AAGTTCAACAAACCCTTTGTCTTCTTAATGATTGAACAAAATACCAAGTCT 1 TTCAAGTTGTTTGGGAAACAGAAGAATTACTAACTTGTTTTATGGTTCAGA CCCCTCTTCATGGGAAAAGTGGTGATCCCACCCAAAAAATAACTGCCTCTG GGGGAGAAGTACCCTTTTCACCACTTAGGGTGGGTTTTTTATTGACGGAGAC GCTCCTCAACCCCTCCCCTCCATCCCTGGCCCCCTCCCTGGATGACATTA 1 CGAGGAGTTGGGGAGGGGAGGTAGGGACCGGGGGAGGGACCTACTGTAAT AAGAAGGGTTGAG Al-,! 1 - TTCTTWCAACT EXPERIMENTAL PROCEDURE 1329 A plasmid containing a partial cD11A insert coding for the carboxy terminus of human aIAT containing nucleotides 1020-1340 as defined by Cohan et al (DI;A, 3, 327-330, 1984) was digested to completion with the restriction enzyme Alu I under conditions as recommended by the manufacturers (Boehringer Mannheim).

Oligonucleotides, 5'd(CCCATGTCTATCCCCCCCk-) (oligo 4).

5'd(CCAAGTCTCCCCICTICATGGG) (oligo 6), and 5'd(,','-'CCC-iCCCCTCCA'LCCCTGG'C'-'CC-C) (oligo tu nucleotides 1141-1159, 1208-1229, and 12751-1302-1 respectively were 51phosphorylated in separate reactions using 1OOnM of oligonucleotide and a two fold excess of 51 Y 32 P adenosine triphosphate (51y 32 P ATP) in buffer containing 50 mM Tris/HCl (pH 9.0), 10 mM MgC12, 20mM DTT, O.ImM spermidine and 0.1 mM EDTA. The specific activity of the 51y 32 P ATP was 5000 Ci/mmol. Phosphorylation was achieved using polynucleotide kinase (1.5 units per pmol oligonucleotide). Each reaction was incubated at 371C for 275 minutes then heated at 100'C for 5 minutes.

Oligonucleotides were purified by polyacrylamide gel electrophoresis and electroelution followed by ethanol precipitation. Phosphorylation was confirmed by autoradiography. % Oligonucleotides, 5'd(GGGGCCATGTTTTTAGAGGCCA), (oligo 3), 5'd(CCTTTGTCTTCTTAATGATTGAACAAAA) (oligo 5), and 5,d(CACCCAAAAATAACTGCCTCTCGCTCCT) (oligo 7), complementary to nucleotides 1117-1138, 1178-1205, and 1245-1272 respectively were also used.

Oligonucleotides 3 and 4 are separated by the dinucleotide 51d(TA) complementary to the dinucleotide 31d(AT) in the human aIAT gene.

Similarly oligonucleotides 5 and 6 are separated by the dinucleotide 5'd(TA). Oligonucleotides 7 and 8 are separated by thE dinucleotidE 51d (CA) complementary to the dinucleotide 3fd(GT) in the human a IAT gene.

The template for the fill in and ligations was an&JAT cDNA plasmid cleaved wth Alu I (Alu I cleaves neither between each pair of oligonucleotides (i.e. 3 and 4, 5 and 6,-or 7 and 8) nor within the sequence of any of the oligonueleotides). 20 pg of the plasmid was incubated in medium salt buffer (supplied by BCL) with the restriction enzyme Alu 1 (17.5 units) for 160 minutes at 37'C. The reaction was shown to be complete by agarose gel electrophoresis on a 1.4% gel in Tris/borate buffer containing 0.5 pg/ml ethidium bromide and visualising under u.v. light.

The following sets of oligonucleotides were annealed to the cD!,A template followed by treatment with pairs of nueleotide triphosphates (dNTPs) as indicated below.

- 29 f Reaction Oligonucleotides 1 3 + 4 C + A 2 3 + 4 T + A 3 5 + 6 C + A 4 5 + 6 T + A 7 + 8 C + A 6 7 + 8 T + A 7 3 + 4 + 5 + 6 C + A 8 3 + 4 + 5 + 6 T + A 9 3 + 4 + 5 + 6 + 7 + 8 C + A 3 + 4 + 5 + 6 + 7 + 8 T + A Each reaction was carried out according to the following protocol:

Reaction buffer: 67 mM Tris/HCl (pH 8.8), (16.6 mM (NH 4)2S04, 6.7 mM MgC1 2' 1OmM 2-mercaptoethanol and 6.7 PM EDTA.

cDNA template: 50 fmol/reaction.

51 32 P oligonucleotide, (4,6 or 8): 300 fmol/reaction.

Non-labelled oligonucleotide (3,5 or 7): 75 fmol/reaction dNTP: 40nmol/reaction (reaction volume 20pl) Step (a) Denaturation of cDNA template at 91'C for 2 minutes.

(b) Annealing of relevant oligonucl'totides at 60'C fo-minutes, (c) Addition of Taq polymerase (0.1 units), (d) Addition of dNTPs, allow to react for 2 minutes at C'C, (e) Flash chill at -70'C for 20 minutes, then allowing mixture to thaw at ambient temperature, (f) Addition of 2.5 jil 10x ligase buffei:

(66C! Tris ' 'HCI (p!-,' 7i.Ci, 6- 7.'.

ml,', DTI, 4 ATP) 1 (g) Addition of T4 DNA ligase (2.5 pl, 0.125 units, previously diluted in 1X ligase buffer), (h) Incubation at 23'C for 40 minutes followed by incubation at 4C for 40 minutes, followed by incubation at 60'C for minutes, (i) Addition of medium salt buffer (supplied by BCL) and 0.5 units Alu I restriction enzyme, (j) Incubation at 37'C for 120 minutes, t (k) Removal of 4 P1 into 10 P1 90% formamide 1 mM EDTA and 0.1% bromophenol blue, (1) Denaturation of sample at 90% followed by rapid chilling at WC, (m) Electrophoresis on 15. polyacrylamide gel in Tris/Borate buffer containing 7M urea, (n) Autoradiography of the gel.

Inspection of the autoradiograph showed bands as expected for the polymerase/ligase products i.e. either the starting 32 P oligonucleotide + extension by incorporation of d1JFs (where complementary to template), or longer pr-6ducts derived from selecti-.1E insertion of complementary dNTPs between oligo pairs followed by ligation. Thus the observed products are:

REACTION EXPECTED SIZE(S) OF OBSERVED SIZE(S) OF PRODUCT(S)PRODUCT(S) 1 20--;n as expected 2 44 ditto 3 2 6 k ditto 4 56 ditto 6 28 d i ', t c, REACTION EXPECTED SIZE(S) OF OBSERVED SIZE(S) OF PRODUCT(S)PRODUCT(S) 7 20 + 26 ditto 8 44 + 56 ditto 9 20 + 26 + 60 ditto 44 + 56 + 28 ditto = Extension of 32 P oligonucleotide by dNTPs k -1.

9 2_

Claims (13)

1. A method for detecting the presence or absence of at least one variant sequence in a single test, which method comprises:1) hybridising a detectable first nucleotide probe and a second nucleotide probe to adjacent segments of each desired diagnostic portion of a target base sequence, the nucleotide sequence of the first and second probe being such that where a hybrid of the first and second probes is formed with a diagnostic portion of a target base sequence the first and second probes are separated by a gap, which gap is defined by at least two nucleotides of the target sequence which nueleotides of the normal and/or variant target sequence comprise all but one of the different naturally occurring nucleotides or less; 2) contacting the diagnostic portion of the target base sequence with one or more nucleotide species under conditions enabling complementary hybridisation, the one or more nucleotide species used being such that either the normal or the variant nucleotide is noncomplementary therewith; 3) subjecting any hybrid obtained to link-age; and 4) determining the presence or absence of each variant sequence by detecting the presence or absence of probe(s) characteristic ol- the presence or absence of each variant sequence; the method being effected such that the nucleotides defining the gar, between the first base sequence are selected to permit linkage of the first and second probes in respect or either the normal or the -jaranltarget sequence but not both such sequences.

2. A method as claimed in claim 1 wherein the first, and second nucleotide probes are selected so that where a gap of only a single nucleotide is formed the sole nucleotide defining the gap is neither the normal nor the variant nucleotide to be detected.

3. A method as claimed in claim 1 or claim 2..-nere4-n the firs- and second probes are separated by a gap, which gap is defirted by twc nucleotides of the target sequence.

.I -

4. A method as claimed in any one of claims 1-3 wherein the probe characteristic of the presence or absence of each variant sequence is a single linked probe comprising the first and second nucleotide probes.

5. A method as claimed in any one of the previous claims wherein the presence or absence of more than one variant sequence is detected simultaneously.

6. A method as claimed in any one of the previous claims which includes at least one analysis of a translocation in a target base sequence in which the detectable first nucleotide probe is adapted to hybridise across a region of potential translocation(s) and the non-detectable second nucleotide probe is adapted to hybridise to sequences adjacent to said potential translocation(s), the non-detectable second nucleotide probe carrying one member of a binCing pair.

7 A method as claimed in any one of claims 1-6 wherein a target sequence or diagnostic portion thereof is subjected to hybridisation with more than two oligonucleotide probes followed by hybridisation with nucleotides complementary to the gap(s) of at least two nucleotides between adjacent ends of the oligonucleotide probes that where a complementary target sequence is present a split probe hybrid is obtained, the detectable first nucleotide probe being a terminal oligonucleotide probe which is detectable and the second nucleotide probe being the other terminal oligonucleotide probe which has attached thereto one member of a binding pair, individual other oligonucleotide probe(s) being hybridised separately but adjacently to a contiguous target sequence between the said terminal oligonucleotide probes, and the indi.,.-idual oligonucleotide probes linked to join thdetectable oligonucleotide probe to the oligonucleotide probe having one member of a binding pair attached thereto to form a linked probe hybrid which is then denatured and contacted with the other member of the binding pair whereby the detectable linked probe nuc"-,eot-7de sequence including the said binding pair may be separatec fror.-- other nucleotide sequences.

W - -3 It

8. A method as claimed in any one of the previous claims wherein prior to step 4), the hybridised probe(s) is separated from the target base sequence followed by sequential performance of steps 1), 2) and 3), the aforementioned procedure being optionally repeated at least once, whereby multiple copies of the the probe(s) for detection are obtained.

9. A probe set comprising a first detectable nucleotide probe and a second nucleotide probe hybridisable to adjacent segments of a diagnostic portion of a target base sequence, the nucleotide sequence of the first and the second probe being such that in use they form a hybrid with a complementary target sequence in which the first and second probes are separated by a gap formed by at least two nucleotides of the complementary target sequence which nucleotides of the normal and/or variant target sequence comprise all but one of the different naturally occurring nucleotides or less, the gap being further characterised in that in use one or more nucleotide species may be used to contact the diagnostic portion of the target base sequence such nucleotide species being selectable such that either the normal or the variant nucleotide(s) is non-complementary therewith.

10. A probe set as claimed in claim 9 for the diagnosis of an inherited disease, condition or disposition.

A kit comprising at least one probe set as claimed in claim 9 or claim 10 together with written or printed instructions for use of the kit and appropriate buffer solutions and/or washing solutions.

12. An -in vitro method of diagnosis which comprises the use of a probe set as claimed in claim 9 or claim 10, or the use of a kit as claimed in clairr, 11.

13. A method substantially as hereinbefore described, with reference to the accompanying Example and Figures.

W Published 1990 W.The Patent Office Sa-5 L2r.d__W-_1 E 47, F c C:ayKer.' ER-P 3FIR:: Frin-J by Cra Saleq Brarcl. S V KCnt