EP0943013A2

EP0943013A2 - Detection of chromosomal abnormalities

Info

Publication number: EP0943013A2
Application number: EP97946747A
Authority: EP
Inventors: Niels Pallisgaard; Peter Hokland
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-12-06
Filing date: 1997-12-08
Publication date: 1999-09-22
Also published as: CA2274315A1; WO1998024928A3; WO1998024928A2; JP2001505428A; AU737190B2; AU5187798A

Abstract

The present invention has provided a method for detection of the presence or absence of chromosomal abnormalities which are associated with a condition e.g. leukaemia in a subject and are each defined by at least one characteristic nucleic acid sequence. In general, the method comprises subjecting a sample of nucleic acids to a multiplex molecular amplification procedure. The multiplex molecular amplification procedure comprises the use of at least 7 mutually distinct primers in one single reaction mixture, each of the at least 7 mutually distinct primers defining an end of at least one characteristic nucleic acid sequence, and wherein at least one of the at least 7 mutually distinct primers defines the first ends of at least two characteristic nucleic acid sequences, said at least two characteristic nucleic acid sequences each being defined in their opposite ends by mutually distinct primers selected from the remainder of the at least 7 mutually distinct primers, whereby the number of amplified characteristic nucleic acid sequences which can be detected upon conclusion of the amplification reaction is at least 1/2xn+1, wherein n is the number of the at least 7 mutually distinct primers. In one embodiment, the use of an internal positive standard containing: I) a nucleic acid fragment present in the sample, and II) primers for amplification of a nucleotide sequence of said nucleic acid fragment is incorporated into the procedure.

Description

DETECTION OF CHROMOSOMAL ABNORMALITIES

FIELD OF THE INVENTION

" The present invention relates to methods for detection of the presence or absence of chromosomal abnormalities associated with a condition, notably a malignant neoplastic disease, in a subject and defined by at least one characteristic nucleic acid sequence. The invention further relates to DNA fragments having specific nucleic acid sequences and their use as either cDNA primers or primers in molecular amplification reactions leading to the detection of chromosomal abnormalities. The invention also relates to a kit comprising selected primers for use of detection according to the invention.

GENERAL BACKGROUND

Chromosomal translocations appear to be important events in the development of tumours (especially haematopoietic tumours) and more than 50 different consistently occurring translocations have been described (Rabbitts, T. H. Nature 372:143 (1994)). Many of the chromosomal aberrations have been found to be specific to particular subtypes of leukaemia or lymphoma .

The identification of translocations in haematopoietic malignancies is therefore of great diagnostic and prognostic value. The diagnosis of acute leukaemia is multidisciplinary with standard pathology, immunology and cytogenetics as the most often used methodologies. In this setting, immunopheno- typing using flow cytometry and monoclonal antibodies provide a speedy and accurate differentiation between lymphoid and myeloid lineages, while a bone marrow biopsy delineates the degree of malignant infiltration simultaneously with revealing the extent of remaining normal haematopoiesis . Neither immunophenotyping nor histology seem to be able to provide satisfactory tools for prognosticating the patients. In contrast, cytogenetic evaluation, while being time consuming, has been shown to delineate both patient groups with favourable as well as poor prognosis. The basis for the value of cytogenetics as a prognostic tool is the existence of a number of balanced chromosomal translocations, where unique genetic sequences are created (for review see Rabbitts, 1995) . Cloning of the translocation break points have indicated that these genes can be altered at the level of their expression or in the properties of the encoded proteins. These alterations appear to play an integral role in the development and possibly in the progression of the disease.

Molecular studies of chromosomal rearrangements connected with the development of haematopoietic tumours have provided important insights into the mechanism of tumorigenesis . The translocations may alter the function or activities of cellular proto-oncogenes located at or near the breakpoint. These proto-oncogenes are normally involved in control of cellular growth, differentiation or apoptosis. The oncogenic conversion may occur by two general mechanisms, either (i) by juxtaposition of a cellular proto-oncogene to the regulatory element of a tissue specific gene, e.g. the immunoglobulin and T-cell receptor genes in leukaemia, leading to inappropriate expression of the oncogene (Leder, P. et al . , Science 222:765 (1983); Finger, L. R. et al . , Science 234:982 (1986) ) , or (ii) by creating fusion genes coding for chimeric proteins with functional features different from the wild- type protein (Borrow, A. D. et al . , Science 249:1577 (1990); de The, H. et al . , Nature 347:558 (1990)).

Translocational breakpoints are highly conserved and ge- nerally within the introns of the affected genes. This is properly due to constrains on the reading frame and on protein (mal) function, but also intron size and the presence of repeated (e.g. Alu) sequences or sequences homologous to B- and T-cell specific recombinase recognition sites within the introns may target and influence the frequency of translocations. However a number of fusion-genes have been found in several variant sizes. Sequence analysis has revealed that the reading frame of the fusion protein variants is preserved, and shows that there may be some freedom in the joining of protein domains in the generation of the oncogene.

In both acute myeloid and lymphoid leukemias, one of the genes involved in the fusion is most often a transcription factor which appears to have a direct role in haematopoiesis and which, following the translocation, is frequently fused to a second gene not normally active in haematopoietic cells. In some instances the same gene is involved in fusion with more that one chromosomal partner.

A translocational breakpoint gene may have several fusion partners, the most promiscuous example is the MLL gene at chromosome band llq23, where 10 different fusion partners together with an internal duplication has been described. The MLL/AF4 fusion gene, detected in t(4;ll) (q21;q23) translocations, is only observed in paediatric ALL, whereas the MLL/AF6 fusion gene detected in t(6;ll) (q27;q23) translocations is seen in a subgroup of AML patients (Prasad, R, et al., Cancer Res. 53:5624 (1993)). The t (10; 11) (pl4;q23) translocation, where the MLL is fused to the AF10 gene, has been described in both paediatric ALL and AML patients. Thus depending on fusion partner the MLL gene can contribute to the pathogenesis of either lymphoid or myeloid malignancies or both. A number of breakpoint genes have been found fused to various partners in different translocations. e.g. E2A/PBX and E2A/HLF in t(l;19) (q23;pl3) and t(17;19) (q22;pl3) or PML/RARα. PLZF/RARo;, NPM/RARα, NPM/ALK, NPM/MLF in t (15;17) (q22;q21) , t (11; 17) (q23 ;q21) , t (5 ; 17) (q35 ;q21) , (2;5) (p23;q35) , t (3 ; 5 ) (q25.1 ;q35) , respectively. Thus at least a subset of the translocations detected in haematopoietic malignancies can be grouped into "fusion-gene families" . Identification of translocations has generally been performed with karyotyping by G-banding or more recently by Fluorescent In Situ Hybridization (FISH) . However, chromosome preparation from clinical samples is often not feasible and the cytogenetic based diagnoses are not sensitive to a small fraction of abnormal cell, i.e. are not helpful for monitoring for relapse. Cytogenetic analysis may detect gross aberrations, but not submicroscopic alterations. Some of these problems may be overcome by using PCR based techniques. However, only four different chromosomal abnormalities have been identified by multiplex- PCR, Repp, R. et al; Detection of Four Different llq23 Chromosomal Abnormalities by Multi- plex-PCR and Flourescence-Based Automatic DNA-Fragment Analysis : Leukemia (1995) 9:210-215

In 10-30% of the patients with a normal karyotype a translocation specific fusion gene can be detected by PCR techniques, indicating that a second chromosomal rearrange- ment has occurred restoring the normal karyotype. A PCR analysis is rapid and very sensitive, but will generally only detect one specific fusion-gene. A major drawback of the PCR method is that it is time consuming and that false negatives are difficult to detect. Thus, the high number of diversified translocations, which have hitherto been demonstrated in acute leukaemia, has precluded its use as a screening tool.

For nearly all translocations, where the genes involved have been identified, a PCR based technique for the detection of the fusion-gene has been described. However, due to the variation in reaction conditions and detection systems, the number of fusion-genes, and the amount of patient material needed, it would be almost impossible with the present methods to screen a patient for the fusion-genes described.

Thus, there is a definite need for fast and reliable screen- ing methods which render possible a prognostic evaluation of patients suspected of suffering from e.g. malignant diseases. OBJECT OF THE INVENTION

It is an object of the invention to provide a simplified and reliable method for the detection of families of chromosomal abnormalities (such as translocations) , which do not suffer the drawback of the prior art methods. Accordingly, a fast and safe method resulting in a very specific diagnosis, it is a futher advantage that the method may be performed by use of as small amounts of sample as possible, due the fact that the patients includes children and babies from which even 20 ml blood is a considerable amount. Further, it is an object of the invention to provide means (especially in the form of specific useful primers) for such a novel method.

DESCRIPTION OF THE INVENTION

The inventors of the present invention have provided a method for detection of the presence or absence of chromosomal abnormalities which are associated with a condition in a subject and are each defined by at least one characteristic nucleic acid sequence. In general, the method comprises subjecting a sample of nucleic acids to a multiplex molecular amplification procedure using multiple and mutually distinct primers in one single reaction mixture, wherein each of the primers defines an end of at least one characteristic nucleic acid sequence. The method according to the invention makes it possible, with a minor amount of work, to screen a sample of nucleic acids for a very high number of chromosomal abnormalities which may occur in a subject. By employing the methods of the invention it has become possible to detect about 50 chromosomal rearrangements with more than 80 subtypes, all performed in one single experimental round.

One distinguishing feature of the invention is the use of amplification primers which constitute the one half of more than one pair of primers used in the amplification reaction, thereby reducing the number of primers necessary to carry out the amplification of all sequences of interest.

For instance, if combining the prior art methods, it would be necessary to use two primers for each individual chromosomal abnormality to be detected. The present invention, on the other hand, exploits the fact that e.g. translocations fall within families, wherein one half of one translocation product is present in at least one other translocation product. In the simple situation where two such translocation products should be detected, it would only be necessary to use a total of three primers. In a more complicated setup, where e.g. 5 translocation products (which all share one translocation "half") should be detected, a total number of 6 primers could ideally be used; this is in strong contrast to the 10 primers which would be necessary when performing a multiplex amplification utilizing the prior art primer systems.

Hence, the inventor has utilized the existence of families of translocations so as to reduce the total number of primers necessary to detect virtually all known chromosomal abnormal - ities associated with malignant diseases of haematopoietic origin. In addition, it has been achieved to perform multiplex PCR reactions wherein the number of primers is no less than 7, i.e. an unprecedented high number of primers present in a multiplex PCR for this purpose.

Thus, in a first aspect the invention relates to a method for detection of the presence or absence of chromosomal abnormalities, each of these chromosomal abnormalities being associated with a condition in a subject and each of these chromosomal abnormalities being defined by at least one characteristic nucleic acid sequence, the method comprising

a) obtaining a sample of nucleic acids derived from a subject which may harbour one of said chromosomal abnormalities , b) subjecting the sample of nucleic acids to a multiplex molecular amplification procedure, wherein a number of said characteristic nucleic acid sequences, if present in a sufficient amount, will be amplified,

c) retrieving the product (s) from step b) , and detecting the presence and/or absence of amplified characteristic nucleic acid sequences and thereby the presence or absence of corresponding chromosomal abnormalities,

wherein the multiplex molecular amplification procedure comprises the use of at least 7 mutually distinct primers in one single reaction mixture, each of the at least 7 mutually distinct primers defining an end of at least one characteristic nucleic acid sequence, and wherein at least one of the at least 7 mutually distinct primers defines the first ends of at least two characteristic nucleic acid sequences, said at least two characteristic nucleic acid sequences each being defined in their opposite ends by mutually distinct primers selected from the remainder of the at least 7 mutually distinct primers, whereby the number of amplified characteris- tic nucleic acid sequences which can be detected upon conclusion of the amplification reaction is at least ^xn+1, wherein n is the number of the at least 7 mutually distinct primers .

In order to obtain reliable results from the molecular ampli- fication procedures used in the methods of the invention, it is often necessary to ensure that the molecular amplification has been satisfactorily performed, i.e. to avoid false negative readings upon conclusion of the amplification.

According to the invention, this can be done by amplifying an internal standard (in the form of a nucleic acid fragment) in the reaction mixture together with a set of primers which will initiate and sustain amplification of the standard. Therefore, in another aspect, the invention pertains to a method for detection of the presence or absence of chromosomal abnormalities, each chromosomal abnormality being associated with a condition in a subject and each chromosomal " abnormality being defined by at least one characteristic nucleic acid sequence, the method comprising

a) obtaining a sample of nucleic acids derived from a subject which may harbour one of said chromosomal abnormalities ,

b) subjecting the sample of nucleic acids to a multiplex molecular amplification procedure, wherein a number of said characteristic nucleic acid sequences, if present in a sufficient amount, will be amplified,

wherein the multiplex molecular amplification reaction comprises

1) the use of an internal positive standard containing I) a nucleic acid fragment present in the sample, and II) primers for amplification of a nucleotide sequence of said nucleic acid fragment, and

2) a number, n, of mutually distinct primers each defining an end of a characteristic nucleic acid sequence,

and wherein at least one of the n mutually distinct primers defines first ends of at least two mutually distinct characteristic nucleic acid sequences, said at least two mutually distinct characteristic nucleic acid sequences being defined in their opposite ends by at least two mutually distinct primers selected from the remainder of the n mutually dis- tinct primers, whereby the number of amplified characteristic nucleic acid sequences which can be detected upon conclusion of the amplification procedure is at least xn+1.

When using the phrases "method of the invention" or "methods of the invention" is herein meant the two above aspects of the invention.

It is preferred that the number of primers used in one single reaction mixture is at least 7, but higher numbers are preferred such as at least 8, 10, 12, 14, 16, 20, 26, or at least 30.

It is expected that the number of primers in one single reaction mixture will be at most 50, but in certain situations the number will be at most 40 or even 35.

Preferred specific numbers of primers in one reaction mixture are apparent from the claims .

As used herein, the terms "chromosomal abnormality" and "chromosomal abnormalities" denote chromosomal sequences of nucleic acids which are usually not detectable in normal healthy subjects whereas these sequences are typically found in subjects suffering from diseases, having an increased risk of developing said diseases, or having well-defined chromosomal defects. Typically, such chromosomal abnormalities are translocations, inversions, deletions, duplications .

It follows that the chromosomal abnormality (ies) will normally be associated with a condition such as a disease (often malignant) or a chromosomal defect, or in other words, the chromosomal abnormality will be present in a significantly higher percent of subjects having the condition than in the average population. In this connection, a "characteristic nucleic acid sequence" is a consecutive stretch of nucleotides which is comprised in the genome of a subject having a chromosomal abnormality and usually not in the "average" healthy subject. Further, the characteristic nucleic acid sequence is one the nucleotide sequence of which is uniquely tied to the chromosomal abnormality, i.e. it will not be found in any substantial number of nucleic acid samples from subjects which do not harbour the chromosomal abnormality.

By the term "molecular amplification procedure" is meant a in vi tro procedure in which a nucleic acid sequence is multiplied by use of priming sequences ("primers") which anneal to a target sequence (the "template") and means for initiating and sustaining amplification of the extension products of the primers or complements thereof. Such methods are well-known in the art, but as exemplary can be mentioned the methods described in EP-0 200 362, EP-0 201 184, EP-A-0 368 906, EP- A-0 379 368, EP-A-0 540 693.

When referring to a "multiplex" molecular amplification procedure is, as well-known in the art, meant a molecular amplification procedure which comprises the use of at least three primers and which results in the amplification of at least two target sequences. In general, multiplex molecular amplification procedures are described in a number of patent publications, cf. e.g. EP-A-0364 255.

The preferred molecular amplification method according to the invention is multiplex PCR.

According to the methods of the invention, the primers used in the amplification procedure must be "distinct", by which term is meant nucleic acid primers which are not 100% identical in sequence and which furthermore will not, under the chosen amplification conditions, exhibit substantial mutual competition for annealing to a given target sequence. Important embodiments of the^' methods of the invention are those wherein the sample of nucleic acids is derived from subjects in the form of cDNA. It will be understood that this requires the use of preceding method steps wherein cDNA is provided by employing reverse transcription of mRNA derived from the subjects and such a procedure thus limits the number of characteristic sequences to be detected to those which are actually transcribed in at least some of the subject's cells. On the other hand, the procedures for retrieving mRNA from cells are well-established in the art and involve relatively few problems in a standard setup.

It should be emphasized though, that the methods of the invention are in no way restricted to use of cDNA as template molecules in the amplification procedures. One interesting possibility will be to extract chromosomal DNA from the subject's cells and perform the multiplex molecular amplification either directly thereon or restriction fragments thereof. In this way it will be possible to detect chromosomal abnormalities which do not give rise to an appa- rent phenotype at the time of extraction but which nevertheless may be an important marker for the condition of interest.

It is, though, preferred that the molecular amplification procedure performed in the method of the invention utilises cDNA obtained by use of specific or non-specific cDNA primers in a separate molecular amplification procedure wherein the templates in the procedure are in the form of mRNA derived from the subject.

In standard schemes for obtaining mRNA, the cDNA primers used are normally non-specific, and the mRNA extraction is therefore "randomly" primed. The present inventor has discovered that markedly superior results are obtained in the detection phase when a mixture of specific cDNA primers are used for synthesis of cDNA from total RNA. In fact, the use of speci- fic cDNA primers has given rise to an approximately 25 to 125 fold increase in sensitivity depending on the system in question.

It is therefore especially preferred that the cDNA primers are specific and in fact, the use of specific cDNA primers when coupled to subsequent multiplex molecular amplification procedures is in itself believed to be a novel approach which leads to a substantially higher sensitivity in the multiplex PCR reaction. Thus, another part of the invention is the combination of such specifically primed cDNA production with subsequent multiplex molecular amplification.

By the term "specific" when used in conjunction with cDNA primers is herein meant that the cDNA primers are predesigned to anneal to target RNA sequences which predominantly exist in RNA transcribed from the above-defined characteristic nucleic acid sequences.

According to the invention, the number of cDNA primers is preferably at least 20, such as at least 25, such as at least 30, such as at least 50, at least 100, at least 150, or at least 200.

When using cDNA (or any other source of template nucleic acids) for the multiplex molecular amplification procedure it is highly advantageous to avoid the need for exchange of media between the procedure for obtaining the template nucleic acids and the multiplex molecular amplification procedure. This can, according to the invention be done by ensuring that the conditions for obtaining cDNA (or other template nucleic acids) derived from the subject are compatible with the conditions of the molecular amplification procedure. In other words, the chemical composition of the medium for the molecular amplification procedure in the inventive method should be substantially the same as that of e.g. the cDNA synthesis procedure, whereas the melting point of any residual cDNA primers should be different from the melting point of the primers used in the multiplex molecular amplification procedure. In this way, it will be possible to restrict the manipulation of test tubes to a minimum and thereby avoid contamination of the samples prior to the multiplex molecular amplification procedure.

" A preferred embodiment of the invention is a method of multi- plex molecular amplification, wherein said multiplex molecular amplification is a nested molecular amplification procedure such as a nested polymerase chain reaction. It is well-known that nested PCR enhances the specificity of any PCR reaction by excluding a large number of artefactual amplification products resulting from the initial round of PCR. Suitable nested PCR methods to be used according to the present invention are those described in USP 4,683,195, and especially the procedures described in EP-A-0 519 338, because these further ensures that no change of media or reactants between the individual steps in the nested molecular amplification procedure need be performed.

An important embodiment of the methods of the invention is a method wherein the chromosomal abnormality is the presence of a transcribed fusion gene. As explained above, a number of expressed fusion genes have been identified which are related in a highly significant manner to various malignant diseases of haematopoietic origin and therefore the detection of the presence of such fusion genes provides important and useful information of the prognosis of the subject, since certain of the malignancies are known to be susceptible to specific regimens of treatment. The presence of such a transcribed fusion gene is typically the result of an inversion, a deletion, a duplication, or activation of a proto-oncogene. Said activated proto-oncogene is typically selected from the group consisting of Hox-11 and evi-l and others as presented in

Rabbits 1994, which is incorporated by reference herein.

However, any genetic variant which is predominantly seen in e.g. malignant cells may be detected according to the invention, when the material subjected to the methods of the invention is not cDNA but for instance nucleic acid fragments derived from a chromosomal source. In this way, a gene like c-myc, which is often overexpressed, may be detected and used as an indication of illness.

" Accordingly, preferred embodiments of the methods of the invention are those wherein at least one of the chromosomal abnormalities is associated with a malignant neoplastic condition, especially a systemic neoplastic malignancy, since a relatively large number of these have been shown to be associated with e.g. expressed fusion genes.

According to the invention, such systemic neoplastic malignancies are selected from the group consisting of leukaemia such as acute leukaemia (AL) , chronic leukaemia (CL) , T-cell acute leukaemia (T-ALL) , B-cell acute leukaemia (B-ALL) , T- cell chronic leukaemia (T-CLL) , B-cell chronic leukaemia (B- CLL) , prolymphocytic leukaemia (PLL) , acute undifferentiated leukaemia (AUL) , acute myelogenous leukaemia (AML) , chronic myelogenous leukaemia (CML) , chronic myelomonocytic leukaemia (CMML) , acute promyelocytic leukaemia (APL) , pre-B-ALL, and pro-B-ALL; lymphoma such as Burkitt's lymphoma (BL) , non-Hodgkins lymphoma (NHL) , Hodgkins lymphoma (HL) , follicular lymphoma (FL) , diffuse large cell lymphoma (DLCL) , T-cell lymphoma, B- cell lymphoma; myelodysplasia; and myeloid.

The following chromosomal rearrangements have all been shown to be coupled to malignancies of the haematopoietic system: dup(llq23) (dup exon 5-9/2); dup(llq23) (dup exon 5-9/4); inv(16) (pl3;q22) ; t(l;ll) (p32;q23) ; t (1;19) (q23;pl3) ; t (10;11) (pl4;q23) t(10;ll) (pl4;q23) t (10;11) (pl4;q23) t(10;14) (q24;qll) t (11;17) (q23;q21) 11; 19) (q23;pl3.1) ; 11; 19) (q23;pl3.3) ; 12; 21) (pl3;q22) 12; 22) (pl3;qll) 15; 17) (q21;q22) 15; 17) (q21;q22) 16;21) (pll;q22) 17; 19) (q22;pl3)

3) (p21;q26) ;

5) (p23;q35) ;

21) (q26;q22) ;

3) (q21;q26) ;

5) (q25.1;q34) ;

11) (q21;q23) ;

12) (q33;pl3) ;

17) (q35;q22)

11) (q27;q23) ;

9) (p23;q34) ;

10) (q35;q24) ;

9) (q34;q32) ;

21) (q22;q22) ;

11) (q22;q23) ;

12) (q34;pl3) ;

22) (q34;qll)

11) (ql3;q23) ; and tall^d1"3 (40 kb deletion) ,

(The letter "t" used in such indications of chromosomal rearrangements denotes a translocation, "inv" denotes an inversion", and "dup" a duplication.)

In connection with these abnormalities, the following genes often become expressed: CBF/3/MYH11, SIL1/TAL1, MLL1, EVI-1, MLL1/AFX1, MLLl/AFlp, MLLl/AFlq, E2A/PBX1, E2A/HLF, EVI1, NPM/ALK, NPM/MLF, AMLl/EVIl, MLL1/AF4, TEL/PDGf/3, NPM/RARo;, DEK/CAN, SET/CAN, MLL1/AF6, HOX11, AML1/MTG8, MLL1/AF9, BCR/ABL, MLL1/AF10, MLL1/AF17, PLZF/RARα, MLL/ELL, MLL/ENL, TEL/AML 1, PML/RARα, FUS/ERG, AMLl/MDS, AMLl/EAP, TEL/MN1, MLL exon 5-9/2, and MLL exon 5-9/4.

The above are thus the most preferred chromosomal abnormal- " ities and genes which are detected in the methods of the invention, and primers (both cDNA and PCR) used in the methods of the invention should therefore be designed in order to specifically amplify sequences characteristic of these rearrangements.

Even though the main body of knowledge of the correlation between chromosomal rearrangements and malignancies is confined to the tumours of the haematopoietic system, knowledge do exist of similar correlations in other, solid tumours, cf, Rabbits, 1994. The methods of the invention are therefore also used to detect neoplastic conditions which are non- systemic neoplastic malignancies. Exemplary of such malignancies are non- systemic neoplastic malignancies selected from the group consisting of carcinoma, adenocarcinoma, liposarcoma, fibrosarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, glioma, neuroblastoma, medullablastoma, malignant melanoma, neurofibrosarcoma, heamangiosarcoma, lymphangiosarcoma, malignant teratoma, dysgerminoma, seminoma, and choriocarcinoma. When the neoplastic disease is carcinoma it is preferably selected from the group consisting of carcinoma of the breast, bronchus, colorectum, stomach, prostate, ovary, lymphoid tissue, lymphoid marrow, uterus, pancreas, oesophagus, urinary bladder, kidney, or skin.

Especially interesting malignant neoplastic conditions are selected from the group consisting of papillary thyroid carcinoma, Ewing's sarcoma, liposarcoma, rhabdomyosarcoma, synovial sarcoma, and melanoma of soft parts, since all of these are positively associated with genomic rearrangements, cf. Rabbits 1994. The sample of nucleic acids ^"used in the methods of the invention is typically derived from cells of the bone marrow in the subject or from peripheral blood cells in the subject. This is especially interesting in the cases wherein the disease to detect is a malignant disease of the haematopoietic system, but also conditions characterized by chromosomal defects (e.g. Downs syndrome) may be detected this way. However, especially for the purpose of detecting the above-mentioned chromosomal defects, the sample may be derived from any other source in the subject, but interesting origins are placental cells, foetal cells, and amniotic fluid. A sample of 5 million mononuclear cells will normally be sufficient to deliver an amount of nucleic acid of 5 μg (between 3 and 8 μgr) RNA, however it depends on the growth rate of the cells. A 20 ml sample from the bone marrow generally corresponds to 5 μg (between 3 and 8 μg) RNA. Accordingly, as little as 10 ml of the sample may be sufficient for the method according to the present invention

In order to facilitate detection of amplified characteristic sequences, at least one of the primers used in the multiplex molecular amplification procedure may according to the invention be labelled. The label can be a radioactive label, a coloured label, a fluorescent label, a biotinyl group, an enzymatic group, a phosphate, an amin a tiol, or any other moiety which can be detected directly or indirectly. For instance, a biotinyl group may in itself be labelled, but it is also possible to detect the presence of the biotinylated nucleic acid fragment by reacting the mixture with labelled avidin or streptavidin.

In especially preferred embodiments of the methods of the invention, the primers are labelled with a fluorescent label or a coloured label. By using primers in the multiplex molecular amplification procedure which are both labelled and unlabelled it also becomes possible to use the degree of fluorescense in a quantitative way. If, for instance, a primer exists in two versions which are different labelled e.g., a labelled and an unlabelled, and is used in differendt known amounts the amplification products will exhibit an average degree of labelling which correspons with the initial defined ratios of label due to the stochastic distribution of the labels in the amplified product. Hence, even though relatively few fluorescent labels of primers are known, it is possible to detect a large number of different amplified fragments when the above- indicated technique is used.

Instead of using labels it is of course possible to carefully choose primers which pairwise gives rise to amplified products of different lengths. The presence or absence of amplified products is then detected by use of various methods which are able to detect the amplified fragments on basis of their size/sequence, methods such as gel electrophoresis, sequence analysis, HPLC, FPLC, flouresence spectofotometri and other suitable chromatograhphic methods.

Alternatively, a labelled means for detecting the amplified products may be used, such as other nucleic acid fragments which will hybridize to the product and thereafter be detect- able by virtue of the label. Such methods are well-known in the art .

Even though it according to the invention is possible to use a large number of primers in the same reaction vessel, there is an upper limit beyond which the amplification procedure becomes too unstable and unreliable. In an important embodiment of the invention, wherein the sample of nucleic acids is subjected to at least two multiplex molecular amplification as defined herein, i.e. the sample (s) derived from the subject is split into several aliquots which each are subjected to a multiplex molecular amplification procedure according to the invention. It is preferred (in view of the reduced time consumption) that the at least two multiplex molecular amplification procedures are carried out in parallel, and it is especially preferred that the at least two multiplex molecu- lar amplification procedures are carried out under substan- tially the same conditions with respect to physical parameters and timing; the latter preferred embodiment has the advantage of allowing the use of e.g. the same reaction buffers (with the exception of primers) and the same thermo- ^" cycling scheme for all aliquots. In essence, all reactions are thus performed in the same thermocycler.

The exact number of different amplification procedures the sample is subjected to may vary, but is preferably at least 3, such as at least 4, 5, 6, 7, 8, 9, 10, 11, 12, or at least 15. Higher numbers may be necessary, depending on the number of families of chromosomal abnormalities need be detected.

The internal standard used in one of the methods of the invention is preferably a cDNA molecule derived from the subject and most preferably said cDNA molecule is obtained by use of specific or non-specific cDNA primers in a molecular amplification procedure wherein the templates in the procedure are in the form of mRNA derived from the subject.

In fact, by deriving the internal standard from the very cells which are the source of the sample of nucleic acids derived from the subject and by using the same type of steps to obtain the internal standard as the ones used for obtaining the nucleic acids derived from the subject, a reliable indication is obtained of the correct execution of all steps leading up to and including the multiplex molecular amplifi- cation procedure. The demonstration of the presence of the internal standard in the final mixture of amplified products will in such a setup indicate that all previous steps have been performed satisfactorily, whereas the demonstration of the absence of the standard will indicate that the assay should be repeated.

Hence, in the case where the sample of nucleic acids derived from the subject is constituted of cDNAs, the internal standard is also cDNA which has been obtained in parallel to the other cDNAs, including the molecular amplification procedure leading to the provision of the other cDNAs .

However, less reliable but nevertheless satisfactory confir- " mation of the correct execution of the various process steps may for instance be obtained by one of the following schemes:

1) A RNA fragment of known sequence is added to the total RNA mixture from which mRNA is extracted, whereafter the subsequent products of the known RNA are obtained from reverse transcription PCR; in this way, all steps but the total RNA extraction are confirmed;

2) A nucleic acid fragment of known sequence is added to the multiplex molecular amplification mixture together with appropriate primers; in this way the multiplex molecular amplification procedure in itself can be confirmed.

It is however preferred to use the internal standard "all the way" and in such embodiments of the invention, a cDNA molecule constituting the internal standard corresponds to a constitutively expressed RNA fragment. In the present examples is used a sequence from the constitutively expressed gene E2A, but any gene which is constitutively expressed in the cells of interest may be used as target. In embodiments where the nucleic acids constituting the sample are derived directly from chromosomal DNA, any "normal" gene sequence may be employed as internal standard.

A crucial factor in the inventive methods are the choice of primers used in the multiplex molecular amplification procedure. In general, the primers are designed using standard software known to the skilled person, and a number of criteria must be met by the primers in the reaction mixture:

1) primers must hybridize to their respective target sequences at or below substantially the same temperature, preferable within a temperature diffenrence of 5°C (they should have the same melting point) ; in the setup reported in the following examples, the melting point has been chosen to be approximately 70°C,

"2) primers must be substantially specific for their respec- tive target sequences, meaning that they will not initiate polymerization from other template sequences than "their own" and that they are not capable of hybridizing with each other. This specificity is obtained by the fact that the primers are completely complementary to the target sequence, however up to 3 point differencies (mutations) may still result in a specific priming.

3) primers should exhibit substantially no intramolecular hybridization, or in other words, there must be a minimum of secondary structure in each primer, that is normally the case when the delta G is above -1 within the primer,

4) primers must have a higher melting point in the 5' -end than in the 3' -end, i.e. they have a high internal stability in the 5' end and a relatively low stability in the 3' end, the difference in melting point is preferable above 1°C, such as above 2°C, preferable above 3°C, such as above 4°C and more preferred above 5°C, such as above 6°C, howver, the exact difference may also depends on other desired properties of the primers .

5) no two primers are, in the molecular amplification pro- cedure, capable of together initiating and sustaining amplification of nucleic acid fragments in the sample which correspond to normally occurring sequences not associated with a condition in the subject,

6) no primer should preferably contain more than 5 consecu- tive guanidyl residues, such as not more than 3 guanidyl residues . 7) they should exhibit substantially no intermolecular hybridization, which may be obtained for the primer dimer having a delta G being above -10.

A further restraint on the choice of primers is that they should pairwise give rise to fragments of different lengths when the molecular amplification procedure is the one resulting in the amplified products to be detected (i.e. the last molecular amplification procedure, e.g. the second PCR in a nested PCR) and the procedure is one wherein the amplified products are distinguished by their length/sequence. In such a situation the amplified fragment should typically have a length of between 100-400 bp.

It will be understood that the precise choice of primers can be varied in an almost indefinite number of ways as long as they conform with the sequences of e.g. the fusion genes to be detected and the above criteria are met. However, as PCR primers the primers having any of SEQ ID NOs 33-177 are currently being especially preferred. As cDNA primers, the primers having any of SEQ ID NOs 1-32 and 178-182 are cur- rently being preferred.

In a further aspect, the invention also relates to a kit comprising 7 mutually distinct primers. The kit may comprise any disired combination of primers for the methods for detection of the presence or absence of chromosomal abnormalities according to the present invention. Accordingly, the kit may comprise primers selected from the group of cDNA primers consisting of SEQ ID NO: 1 through SEQ ID NO: 32 and SEQ ID NO: 178 through SEQ ID NO: 182 and of PCR primers selected from SEQ ID NO: 33 trough SEQ ID NO: 177. The kit according to the present invention may also comprise additives such as buffers, enzymes, and stabilizing agents known in the art.

In a preferred embodiment, the primers are attached to a surface of a device such as a well, e.g of a multiplate, a cappilary tube, a stick, or a bead (such as a magnetic bead) . In this connection, the primers may be dried or in other any suitable form including being contained in a polymer vehicle. In a further embodiment, the primers in the kit are in a liquid form contained in e.g. a tube or well.

The above-mentioned specific primers also in themselves constitute another aspect of the invention as do homologues thereof which will perform equally effectively in the PCR amplifications described herein or as cDNA primers.

LEGENDS TO FIGURES

Figure 1. Setup of the multiplex PCR amplification reaction. The figure shows representative results of gel electrophores- es of nucleic acid samples from one patient subjected to 8 multiplex nested PCR amplifications, each multiplex nested PCR using the primer mixes 1-8. The upper lane (lane 1) represents molecular weight markers. The band which is present in lanes 1-8 is the internal standard, while the second band in lane 5 (primer mix 4) is a detected chromosomal abnormality as highlighted in bold.

Figs. 2A and 2B. The figure shows gel electrophoreses of nucleic acid samples from 18 individual patients, subjected to multiplex nested PCR amplification using the primer mixes 1-8. In each patient one or more chromosomal abnormalities has been detected by the PCR giving rise to a typical and readily identifiable pattern of bands. Above each panel, representing one patient, the actual chromosomal abnormality has been specified.

Figure 3. The figure shows samples positive in the multiplex analysis reanalyses with individual primer sets. The upper three panels each represent gel electrophoreses of multiplex nested PCR performed on nucleic acid samples from one patient. From each of these gels one lane shows that the individual harbours a chromosomal abnormality, the precise nature and variant of which cannot be readily determined. In order to clearly identify this abnormality nucleic acids from each patient were subjected to another round of PCR (lower three panels) this time using individual primer sets able to determine the nature of the fusion genes involved.

EXAMPLE 1

Detection of chromosomal abnormalities in patient samples using multiplex PCR

MATERIALS AND METHODS

Patient Samples and Cell Lines

Bone marrow or peripheral blood samples were fractionated on a Ficoll gradient and cryopreserved before use. The cell lines Karpas-299, ML-2, Mono-Mac-6, NB-4, 697, JOSK-I, NALM-6 and RPMI-8402 were obtained from DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) - (DSM accession numbers 31, 15, 124, 207, 42, 155, 128 and 290 respectively). The cell lines RS4;11 and MV-4-11 were obtained from the American Type Culture Collection (Rock- ville, MD) (ATCC accession numbers CRL 1873 and CRL 9591, respectively) . The cell line HAL- 01 is described in Ohyashiki et al . (1991), Leukaemia 5: pp. 322-331. The cell lines were all cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. The medium for the cell line Mono-Mac-6 was supplemented with 9 μg/mL bovine insulin.

RNA Preparation

Total RNA was prepared either by the guanidinium thiocyanate- phenol chloroform method [Chomczynski, Anal. Biochem 162:156, 1987] or by using a RNeasy Kit (Quiagen) according to the manufacturer's instructions. The RNA solution was subsequently treated with 0.1 unit/μL RNase-free DNase (Boeh- ringer) in 50 mmol/L Tris-HCl, pH 8.0 , 10 mmol/L MgCl₂ at

37°C for 30 minutes. After the DNase treatment EDTA, pH 8.0 was added to 10 mmol/L and the RNA solution was extracted once in phenol/chloroform 1:1, sodium-acetate added to 200 mmol/L and precipitated with 1 volume of isopropanol. The RNA was pelleted in an eppendorf centrifuge at 13.000 rpm for 30 minutes and washed with 80% ethanol. The RNA was resuspended in 25 μL DEP ddH₂0 and 5 μL withdrawn for quantification on a Genequant (Pharmacia) . Subsequently the RNA was diluted to 0.1 μg/μL and stored until use at -80°C in 10 μL aliquots.

Reverse Transcriptase PCR

One microgram of total RNA was incubated for 5 minutes with a mixture of translocation-specific-cDNA-primers (2.5 pmol of each) and then reverse transcribed to cDNA by incubation at 37°C for 45 minutes in a total volume 25 mL containing 20 units RNase inhibitor (Boehringer) , 1 mmol/L of each dNTP, 10 mmol/L dithiothreitol , 1XRT buffer (50 mmol/L Tris-HCl pH 8.3, 75 mmol/L KC1 , 3 mmol/L MgCl₂) , and 400 units Moloney murine leukaemia virus reverse transcriptase (BRL, Bethesda, MD) . At the end of the incubation, the cDNA reaction mixture was diluted with ddH₂0 to 50 μL. The PCR amplification was performed as 8 parallel nested (two round) multiplex reactions in a Perkin Elmer 9600 thermocycler. Five μL of diluted cDNA reaction was added to each of eight 20 μL multiplex mixtures which contained 1.1 X PCR buffer (10 mmol/L Tris-HCl pH 8.3, 50 mmol/L KC1 , 1.5 mmol/L MgCl₂) , 0.2 mmol/L of each dNTP, 12,5 pmol of each primer and 1.5 unit AmpliTaq-Gold polymerase (Perkin Elmer) . The first PCR reaction time consisted of an initial activation of the polymerase at 95°C for 15 minutes, followed by 25 cycles of PCR amplification (annealing at 58°C for 30 seconds, elongation at 72°C for 1 minute, and denaturation at 95°C for 30 seconds) . After the first round of PCR, a 1 μL aliquot from each of the 8 PCR reactions was transferred to eight 24 μL second round multiplex mixtures which contained 1 X PCR buffer (10 mmol/L Tris- HCl pH 8.3, 50 mmol/L KC1 , 1.5 mmol/L MgCl₂) , 0.2 mmol/L of each dNTP, 5-12.5 pmol of each primer and 1.5 unit AmpliTaq- Gold polymerase. The second PCR reaction time consisted of an initial activation of the polymerase at 95°C for 15 minutes, followed by 20 cycles of PCR amplification (annealing at 58°C for 30 seconds, elongation at 72 °C for 1 minute, and denaturation at 95°C for 30 seconds) followed by a 10 minutes extension at 72°C. Fifteen μL of the PCR reactions were electrophoresed in a 1.5% agarose gel for 60 minutes at 100V and stained with ethidiumbromide . Positive samples were reanalyzed to verify/determine the translocation (s) by performing cDNA and nested PCR with the individual primer sets using the same conditions as for the multiplex PCR except that only 0.75 unit/reaction of AmpliTaq-Gold polymerase was used. Positive samples with limiting amount of RNA were reanalyzed by performing only the second round of PCR with the individual primer sets using 1 μL from the first round multiplex PCR as template. This analysis was performed with and without the internal control primers and translocations were confirmed by DNA sequence analysis. Negative controls without DNA template were included for all PCR reactions mixtures. To minimize the risk of contamination filtertips were used in all steps and four different laboratories with indigenous pipettes were used for the preparation of stock solutions, the RNA preparation and cDNA synthesis/setup of first PCR, the first to second PCR transfer, and the gel electrophoresis .

Primer design and DNA sequencing

All PCR oligonucleotide primers were designed using the Windows primer analysis software OLIGO version 5.0 (National Biosciences Inc., Plymouth, MN) using data from the EMBL DNA database. Oligonucleotide primers were supplied HPLC purified from DNA Technology, Science Park, DK-8000 Aarhus . DNA sequencing was performed on agarose gel purified PCR fragments using a Taq DyeDeoxy Terminator Sequencing kit (Perkin Elmer) on an automated 373A DNA sequencer (Applied Biosysterns, Foster City, CA) . Both strands of the PCR fragments were sequenced. The cDNA primers were: 1. 12-mer: CBFBMYHA: 1752L12 ,

5' AgC TgC TTg ATg 3' (SEQ ID NO: 1).

2. 12-mer: CBFBMYHA: 1033L12 ,

5' CTg CTg ggT gAg 3' (SEQ ID NO: 2). '3. 12-mer: ALL1AF1 : 4220L12 ,

5' ATg ggA gCT CAg 3' (SEQ ID NO: 3).

4. 12-mer: ALL1AF1Q: 4355L12 ,

5' Agg gCT TTT gAg 3' (SEQ ID NO: 4).

5. 11-mer: E2APRL: 764L11, 5' CCC TCC AgA Ag 3' (SEQ ID NO: 5).

6. 12-mer: ALKNPM: 714L12 ,

5' CAg CgA ACA ATg 3' (SEQ ID NO: 6).

7. 12-mer: AMLIEVI : 2459L12 ,

5' CCC ATC CAT AAC 3' (SEQ ID NO: 7) . 8. 12-mer: ALL1AF4 : 4349L12 ,

5' TTC CTT gCT gAg 3' (SEQ ID NO: 8).

9. 12-mer: TELPDGF: 1003L12 ,

5' CTg CAg gAA ggT 3' (SEQ ID NO: 9).

10. 12-mer: DEKCAN: 1446L12 , 5' TTg gCT ggT ACT 3' (SEQ ID NO: 10).

11. 12-mer: ALL1AF6 : 4150L12 ,

5' CCg ATC ATC TTT 3' (SEQ ID NO: 11).

12. 12-mer: AML1MTG8 :2460L12 ,

5' gTg CgA ACT CTT 3' (SEQ ID NO: 12). 13. 12-mer: AF9:1536L12,

5' CTg CCA TCA CTT 3' (SEQ ID NO: 13) .

14. 12-mer: ALL1AF9 : 4184L12 ,

5' gCA TCC AgT TgT 3' (SEQ ID NO: 14).

15. 12-mer: ABL:797L12, 5' gCT gCC ATT gAT 3' (SEQ ID NO: 15).

16. 12-mer: ALLAF10A: 3997L12 ,

5' CCA Ctg CCT CTC 3' (SEQ ID NO: 16).

17. 12-mer: AF10 : 1150L12 ,

5' ACC TgA gCT gTg 3' (SEQ ID NO: 17) . 18. 13-mer: AF10:750L13,

5' gTA gCC ACA gTA T 3' (SEQ ID NO: 18) .

19. 11-mer: AF17 : 1965L11,

5' gAC ACC ggA Ag 3' (SEQ ID NO: 19) . 20. 12-mer: BCR1 : 2094L12 ,^*

5' Cgg TCg TTT CTC 3' (SEQ ID NO: 20).

21. 12-mer: ALL1ENL: 4271L12 ,

5' TCT CCA CgA AgT 3' (SEQ ID NO: 21). ' 22. 12-mer: ALLELL: 4341L12 ,

5' CCA gCC TTg ATg 3' (SEQ ID NO: 22).

23. 12-mer: EWSERG: 1071L12 ,

5' TgT Agg CgT AgC 3' (SEQ ID NO: 23).

24. 12-mer: E2AHLF: 1726L12 , 5' ggC CTC ATA CTT 3' (SEQ ID NO: 24).

25. 11-mer: E2A:1960L11,

5' GCT TCG CTC AG 3' (SEQ ID NO: 25).

26. 12-mer: AMLIEVI : 4905L12 ,

5' TAA ggC TgC TCT 3' (SEQ ID NO: 26). 27. 12-mer: TELAML1 : 1365L12

5' Cgg Tag CAT TTC 3' (SEQ ID NO: 27).

28. 11-mer: TAL1:263L11,

5' Ccg TcC CTC TA 3' (SEQ ID NO: 28) .

29. 12-mer: AFX1:70L12, 5' Aag TgC CAA Cag 3' (SEQ ID NO: 29) .

30. 11-mer: HOX11 : 897L11,

5' TgC TgC CTC TC 3' (SEQ ID NO: 30).

31. 13-mer: ALL1:417L13,

5' TTT ggT CTC TgA T 3' (SEQ ID NO: 31) . 32. 12-mer:MLFl:320L12

5' Tgg TCT ggA Aag 3' (SEQ ID NO: 32) .

178. ENL:1405L12

5' GCCTGACACCTT 3' (SEQ ID NO: 178).

179. ALL1:3275L11 5' CTGCCCACACC 3' (SEQ ID NO: 179).

180. MN1:5065L12

5' GCCACTAAGCAG 3' (SEQ ID NO: 180).

181. EAP:1012L13

5' TAATCCTCGTCTT3 ' (SEQ ID NO: 181). 182. AMLIEVI: 2820L13

5' GTCCTCTTCAACC 3' (SEQ ID NO: 182). The following tables recite ^'the composition of the 16 different reaction mixtures used in the 8 nested PCR reactions, the interrelationship between the PCR primers and the chromosomal rearrangement to be detected, and the resulting PCR fragments ("NP" mixes are stock solutions adapted for detection of one single rearrangement, whereas "R mixes" are the combinations of NP mixes used in the multiplex nested PCR reactions, finally, the M-mixes are an alternative to the R-mixes, the difference is that the M-mixes detects 5 additional fusion genes, but not the activiation of HOXll and EVYl as tested for in the R-mixes) . The control primer set (NP-mix No. 41) is included in the tests as shown.

R-mix no. PCR Primers, SEQ ID NO: PCR mix PCR Primers SEQ ID NO: PCR mix

1st PCR pmol/μl 2nd PCR pmol/μl

CBFBMYHC:267U22 81 5 CBFBMYH:344U21 129 12.5

CBFBMYHC:752L22 39 5 CBFBMYHC:595L19 125 12.5

CBFBMYHA:919L24 58 5 CBFBMYHA:868L20 124 12.5

ALLAF10A:3730U20 49 5 AL 1AF4:3751U20 114 12.5

ALL1:3955U24 36 5 ALL1:3996U24 88 12.5

Rl AFX1:29L20 68 5 AFX1:5L24 118 12.5

ALL1AF6:4074L21 50 5 AL 1AF6:4037L22 109 12.5

ALL1ELL:4236 23 34 5 ALL1ELL:4191L22 132 12.5

E2APRL:220U21 78 5 E2A:1173U19 103 7.5

E2A:1883 22 82 5 E2A:1844L19 90 7.5

H₂0 H₂0

A LAF10A:3730U20 49 5 ALL1AF4:3751U20 114 12.5

ALL1:3955U24 36 5 ALL1:3996U24 88 12.5

ALL1AF1:4048L22 65 5 ALL1AF1:4031L21 130 12.5

AF17:1937 21 51 5 ALL1AF17:4009L23 119 12.5

ALLAF10A:3968L23 56 5 ALLAF10A:3932L21 134 12.5

R2 ALLAF10B:4031L22 79 5 ALLAF10B:3996L23 131 12.5

AF10:728L22 76 5 AF10:685L21 115 12.5

ALL1:391L23 80 5 ALL1:313L28 110 12.5

E2APRL:220U21 78 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂θ H₂0

E2APRL:220U21 78 5 E2APRL:673L21 95 12.5

E2APRL:696L18 61 5 E2A:1173U19 103 12.5

SIL:24U19 52 5 SIL:83U20 98 12.5

TAL1:203L21 55 5 TAL1:179L20 86 12.5

R3 E2AHLF:1685L20 62 5 E2AHLF:1543L20 100 12.5

TE AML1:871U23 44 5 TELAML1:944U23 104 12.5

TELAML1:1335L23 37 5 TELAML1:1216L21 87 12.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂θ H₂0

AMLIEVI: 1897U21 60 5 AML1MGT8:1895U20 128 12.5

AML1EV 2375L24 53 5 AML1EVI:2345L21 97 12.5

HOX11:590U20 67 5 H0X11:617U22 106 12.5

H0X11:857L21 70 5 HOX11:810L19 113 12.5

AML1MGT8:2437L23 73 5 AML1MGT8:2226L22 102 12.5

R4 TLSERG:649U19 69 5 TLS:690U19 93 12.5

EWSERG:979L22 72 5 TLSERG:945L19 116 12.5

E2APRL:220U21 78 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂0 H₂0 R-mix no. PCR Primers, SEQ ID NO: PCR mix PCR Primers SEQ ID NO: PCR mix

1st PCR pmol/μl 2nd PCR pmol/μl

ALLAF10A:3730U20 49 5 ALL1AF4:3751U20 114 12.5

ALL1:3955U24 36 5 ALL1:3996U24 88 12.5

ALL1AF4:4321L29 57 5 ALL1AF4:4291L25 122 12.5

ALL1AF9:4143L24 48 5 ALL1AF9:4092L24 108 12.5

R5 AF9:1498L22 77 5 AF9:1466L26 105 12.5

ALL1AF1Q:4211L22 64 5 AF1Q:580L20 127 12.5

AL 1ENL:4215L22 42 5 ALL1ENL:4164L19 112 12.5

E2APRL:220U21 78 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂0 H₂0

BCR1ABL:1698U19 54 5 BCR1ABL:1777U19 85 12.5

BCR2ABL:3060U23 59 5 BCR2ABL:3128U22 91 12.5

BCR1ABL:2093L20 83 5 BCR1ABL:2074L23 84 12.5

TELPDGF:309U21 63 5 TELPDGF:343U24 99 12.5

R6 TELPDGF:834L22 45 5 TELPDGF:642L22 94 12.5

E2APRL:220U21 78 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂0 H₂0

DEKCAN:870U24 74 5 DEKCAN:892U21 89 12.5

DEKCAN:1422L21 66 5 SETCAN:925L20 120 12.5

SETCAN:468U23 43 5 SETCAN:552U24 126 12.5

AML1EVI:4331U23 47 5 AML1EVI:4509U21 101 12.5

R7 AML1EVL4866L21 75 5 AML1EVI:4746L25 123 12.5

E2APRL:220U21 78 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂θ H₂0

PLZFRARA:1092U21 46 5 PLZFRARA:1252U21 121 12.5

BCR1:1338U19 40 5 BCR1:1497U21 111 12.5

BCR3:988U19 71 5 BCR3:1057U20 92 12.5

BCR3:1460L19 33 5 BCR3:1428L22 96 12.5

ALKNPM:200U25 35 5 ALKNPM:313U21 117 12.5

R8 ALKNPM:627L21 41 5 ALKNPM:590L19 107 12.5

MLF1:235L27 38 5 MLF1:192L28 133 12.5

E2APRL:220U21 78 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂θ H₂0 NP-mix no. Rearrangement Genes PCR-Primers Nl SEQ ID NO: PCR-Primers N2 SEQ ID NO: PCR Fragment(s) Pos. Controle (1st PCR) (2nd PCR)

CBFp (16q22) CBFBMYHC:267U22 81 CBFBMYH:344U21 129 174 bp

2 inv(16)(pl3;q22) MYHll(16pl3) CBFBMYHC:752L22 39 CBFBMYHC:595L19 125 to Pt 663 bp

CBFβ (16q22) CBFBMYHC:267U22 81 CBFBMYH:344U21 129 544 bp

4 inv(16)(pl3;q22) No MYHll(16pl3) CBFBMYHA:919L24 5'8 CBFBMYHA:868L20 124 337 bp tall"¹-³ SILl(lp34) SIL:24U19 52 SIL:83U20 98 183 bp

45 RPMI

(40 kb deletion) TALl(lp34) TAL1:203L21 65 TAL1:179 20 86 dup(llq23) ALLl(llq23) AH-AF10A:3730U20 49 ALL1AF4:3761U20 114 244 bp

CO 49 Pt CZ dup exon 2-5/8 ALL1:391 23 80 ALL1:313L28 110 CD ALL1 :3955U24 CO 36 ALL1 :39996U24 88

AFXl(X;ql3) ALLAF10A:3730U20 49 ALL1AF4:3751U20 114 235 bp

Cell line:

46 A l(llq23) ALL1:3955U24 36 ALL1:3996U24 88 to t(X;ll)(ql3;q23) Karpas 45

CO AFX1:29L20 65 AFX1:5L24 118 449 bp m m AFlp(lp32) AL AF10A:3730U20 49 AL 1AF4:3751U20 114 187 bp

5 t(l;ll)(p32;q23) ALLl(llq23) AL 1:395BU24 36 ALL1:3996U24 88 to No

ALL1AF1:4048 22 65 ALL1AF1:4031L21 130 301 bp m t AFlq(lq21) AL AF10A:3730U20 49 AL 1AF4:3751U20 114 287 bp

6 t(l;ll)(q21;q23) ALLl(llq23) AL 1:3955U24 36 AL 1:3996U24 88 to No

ALL1AF1Q:4211L22 64 AF1Q:580 20 127 368 bp

PBXl(lq23) E2APRL:220U21 78 E2A:1173U19 95 376 bp Pt+Cell line:

8 t(l;19)(q23;pl3) E2A(19pl3) E2APRL:696L18 61 E2APRL.-673L18 103 697

ALK(2p23) NPMA K:200U25 35 NPMA K_313TJ21 117 302 bp Pt+

9 t(2;5)(p23;q35) NPM(6q35) NPMALK.-627 21 41 NPMALK:590 19 107 Karpas 299 t(3;3)(q21;q26) Activation of AML1EVI:4331U23 47 AM 1EVI:4509U21 101

42 t(2;3)(p21;q26) EVIl(3q26) AML1EVI:4866 21 75 Pt+Cell line: AML1EV 4746 25 123 262 bp t(3;21)(q26;q22)

NP-mix no. Rearrangement Genes PCR-Primers Nl SEQ ID NO: PCR-Primers N2 SEQ ID NO: PCR Fragment(s) Pos. Controle (1st PCR) (2nd PCR)

M F(3q25.1) NPMALK:200U25 35 NPMALK:313U21 117 289 + 333

50 t(3;5)(q25.1;q34) NPM(5q34) MLF1:235 27 38 M F1:192_L28 133 362+406

EVI.l(3q26) AML1EVI:1897U21 60 AML1MGT8:1895U20 128 446 bp Cell line:

11 t(3;21)(q26;q22) AMLl(21q22) AML1EVI:2375L24 53 AML1EVI:2345 21 97 sKm

AF4(4q21) ALLAF10A:3730U20 49 ALL1AF4:3751U20 114 75 bp Cell line:

12 t(4;ll)(q21;q23) ALLl(llq23) ALL1:3955U24 36 ALL1:3996U24 88 to RS4;11

ALL1AF4:4321L29 57 ALL1AF4:4291L25 122 321 bp MV4;11

CO PDGFβ(5q33) TELPDGF:309U21 63 TE PDGF:343U24 99 321 bp cr 13 t(5;12)(q33;pl3)

CD TE (12 l3) TELPDGF:834L22 45 TELPDGF:642L22 94 CO t(5;17)(q35;q22) NPM(5q35) NPMALK:200U25 35 NPMALK:313U21 117 105 bp

51 S-, L-forms RARA(17q21) BCR3:1460L19 33 BCR3:1428L22 96 234 bp m O t(6;9)(_P23;q34) CAN(9q34) DEKCAN:870U24 74 DEKCAN:892U21 89 320 bp m 14 m DEK(6p23) Pt DEKCAN:1422L21 66 SETCAN:925L20 120

3d ?t(9;9)

15 CAN(9q34) SETCAN:468U23 43 SETCAN:552U24 126 393 bp

No m SET(9q34) ■ DEKCAN:1422L21 66 SETCAN:925 20 120

AF6(6q27) AL AF10A:3730U20 49 ALL1AF4:3751U20 114 199 bp

Pt+Cell line:

16 t(6;ll)(q27;q23) ALL(llq23) ALL1:3955U24 36 AL 1:3996U24 88 to ML-2

AL 1AF6:4074L21 50 ALL1AF6:4037122 109 313 bp t(7;10)(q35;q24) Activation of HOX11:590U20 67 H0X11:617U22 106 212 bp

48 Pt+RPMI t(10;14)(q24;qll) HOXll(10q24) H0X11:857L21 70 HOX11:810 19 113

MTG8(8q22) AML1EVI:1897U21 60 AM 1MGT8:1895TJ20 128 353 bp Pt+Cell line:

17 t(8;21)(q22;q22) AMLl(21q22) AML1MGT8:2437 23 73 AM 1MGT8:2226L22 102 Kasumi-1

AF9(9q22) ALLAF10A:3730ϋ20 49 AL 1AF4:3751U20 114 208 bp

18A t(9;ll)(q22;q23) ALLl(llq23) ALL1:3955U24 36 ALL1:3996U24 88 Cell line: to

AF9:1498L22 77 AF9:1466L26 105 322 bp Mono Mac 6

AF9(9q22) AL AF10A:3730U20 49 ALL1AF4:3751U20 114 254 bp

Cell line:

18B t(9;ll)(q22;q23) AL l(llq23) ALL1:3955U24 36 ALL1:3996U24 88 to Mono Mac 6

AL 1AF9:4143L24 48 ALL1AF9:4092L25 108 368 bp

ABL(9q34) ELPDGF:309U21 63 TELPDGF:343U24 99 366 bp

43 t(9;12)(q34;pl3) No EL(12pl3) BCR1ABL:2093 20 83 BCR1ABL:2074 23 84 t(9;22)(q34;qll) AB (9q34) BCR1ABL:1698U19 54 BCR1AB :1777U19 85 320 bp

19 Pt type ela2 BCR(22qll) BCR1ABL:2093L20 83 BCR1AB :2074 23 84

AL l(llq23) ALLAF10A:3730ϋ20 49 ALL1AF4:3751U20 114 t(ll;17)(q23;q21)

22 AF-17(17q21) ALL1:3955U24 36 ALL1:3996U24 88 284 bp No A

AF17:1937 22 51 AL 1AF17:4009L23 119 t(ll;17)(q23;q21) PLZF(llq23) PLZFRARA:1092U21 46 P ZFRARA:1252U21 121 315 bp

23 Pt B RARA(17q21) BCR3:1460L19 33 BCR3:1428L22 96 402 bp

ALLl(llq23) ALLAF10A.-3730U20 49 ALL1AF4:3751U20 114 73 bp t(ll;19)(q23;pl3.3) Pt:

24 ENL(19pl3) ALL1:3955U24 36 ALL1:3996U24 88 to (HB1119 +

ALL1ENL:4215 22 42 ALL1ENL.-4164L19 112 187 bp K0C 33 ect.

ALLl(llq23) ALLAF10A:3730U20 49 ALL1AF4:3751U20 114 217 bp t(ll;19)(q23;pl3.1)

25 ELL(19pl3) ALL1:3955U24 36 ALL1:3996U24 88 to Pt

ALL1EL :4236L23 34 ALL1EL :4191 22 132 421 bp t(12;21)(pl3;q22) EL(12pl3) TELAML1:871U23 44 TE AM 1:944U23 104 293 bp

44 Pt AM l(21q22) TELAML1:1335L23 37 TE AML1:1216L21 87 332 bp t(15;17)(q21;q22) PML(15q22) BCR1:1338U19 40 BCR1:1497U21 111 427 bp Pt+Cell line:

30 type V+L RARA(17q21) BCR3:1460 19 33 BCR3:1428 22 96 NB4

CO

00 t(15;17)(q21;q22) PM (15q22) BCR3:988TJ19 71 BCR3:1057U20 92 393 bp CO 31 Pt type S RARA(17q21) BCR3:1460L19 33 BCR3:1428L22 96

FUS(16pll) TLSERG:649U19 69 TLS:690U19 93 274 bp Pt+ m 32 t(16;21)(pll;q22) ERG(21q22) E 5ERG:979L22 72 TLSERG:945 19 116 (UTP-L12) O z m m H F(17q22) E2APRL:220U21 78 E2A:1173U19 103 390 bp Cell line: —i 33 t(17;19)(q22;pl3) E2A(19pl3) E2AHLF:1685L20 62 E2AHLF:1543 20 100 HAL-01 cz

E2A(19pl3) E2APRL:220U21 78 E2A:1173U19 103 694 bp All Pts. and cell

41 Positive controle ro E2A-.1883 22 82 E2A:1844 19 90 lines en

R-mix Chromosomal genes PCR primers SEQ ID PCR primers SEQ ID PCR Comments no. Rearrangement (1st PCR) NO: (2nd PCR) NO: fragments

Rl inv(16)(pl3;q22) CBFp(16q22) CBFBMYHC:267U22 81 CBFBMYH:344U21 129 174 bp 7 variants

MYHll(16pl3) CBFBMYHC:752L22 39 CBFBMYHC-.595L19 125 to

(NP: CBFBMYHA:919L24 58 CBFBMYHA:868L20 124 663 bp

2,

4, t(X;ll)(ql3;q23) AFXl(Xql3) AL AF10A:3730U20 49 AL 1AF4:3751U20 114 235 bp 3 variants

16, ALL(llq23) ALL1:3955U24 36 ALL1:3996U24 88 to

29,

46) AFX1:29L20 68 AFX1:5L24 118 449 bp

CO t(6;ll)(q27;q23) AF6(6q27) AL 1AF6:4074L21 50 ALL1AF6:4037122 109 199 bp 3 variants

CZ CD ALL(llq23) to O

313 bp t(ll;19)(q23;pl3.1) A l(llq23) ALL1ELL:4236L23 34 ALL1ELL:4191L22 132 157 bp 6 variants m CO ELL(19pl3) to m 421 bp m

R2 t(l;ll)(p32;q23) AFlp(lp32) ALLAF10A:3730U20 49 AL 1AF4:3751U20 114 187 bp 3 variants

ALLl(llq23) AL 1:3955U24 36 A L1:3996U24 88 to rn (NP: AL 1AF1:4048 22 65 ALL1AF1:4031L21 130 301 bp

CD 5, 22, t(U;17)(p23;q21) ALLl(llq23) AF17:1937L22 51 ALL1AF17:4009 23 119 284 bp

21A, A AF17(17q21)

2 IB, t(10;ll)(pl4;q23) AF10(10pl4) ALLAF10A:3968L23 56 AL AF10A:3932L21 134 154 bp 13 variants

21E, type A+B+C+D+E ALLl(llq23) A AF10B:4031 22 79 ALLAF10B-.3996L23 131 to

49) AF10:728L22 76 AF10:685L21 115 389 bp

Dup(llq23) ALLl(llq23) 80 ALL1:313L28 110 244 bp

ALL1:391L23

R3 t(l;19)(q23;pl3) PBXl(lq23) E2APRL:220U21 78 E2A-.1173U19 95 376 bp

E2A(19pl3) E2APRL:696L18 61 E2APRL:673L18 103

(NP: tall""-³ SILl(lp34) SIL:24U19 52 SIL:83U20 98 183 bp 8,

33, TALl(lp34) TAL1:203 21 55 TAL1:179L20 86

44, t(17;19)(q22;pl3) HLF(17q22) E2AHLF:1685L20 62 E2AHLF:1543L20 100 390 bp

45)

CO E2A(19pl3)

CZ CD CO t(12;21)(pl3;q22) TEL(12pl3) TELAML1:871U23 44 TELAML1:944U23 104 293 bp 2 variants

AM l(21q22) TELAML1:1335L23 37 TELAML1:1216L21 87 332 bp Z

— i m R4 t(3;21)(q26;q22) EVI-l(3q26) AML1EVI:1897U21 60 AML1MGT8:1895U20 128 446 bp co AMLl(21q22) AML1EVI:2375L24 53 AML1EVI:2345 21 97 m (NP: m 11 t(7;10)(q35;q24) Activation of HOX11:590U20 67 H0X11:617U22 106 212 bp t(10;14)(q24;qll) HOXll(10q24) H0X11:857L21 70 HOX11:810 19 113 17,

32, t(8;21)(q22;q22) MTG8(8q22) AM 1MGT8:2437L23 73 AML1MGT8:2226L22 102 353 bp

48) AMLl(21q22) co t(16;21)(pll;q22) FUS(16pll) TLSERG:649U19 69 TLS:690U19 93 274 bp

ERG(21q22) EWSERG:979 22 72 TLSERG:945L19 116

R-mix Ch omosoTnftl genes PCR primers SEQ ID PCR primers SE ID PCR Comments no. Rearrangement (1st PCR) NO: (2nd PCR) NO: fragments

Rδ t(4;ll)(q21;q23) AF4(4q21) AI-LAFlQA:3730tJ20 49 A L1AF4:3751U20 114 75 bp 9 variants type A+B+C A Ll(llq23) ALL1:3956U24 36 A L1:3996U24 88 to

(NP: AI_ 1AF4:4321L29 57 AL 1AF4:4291 25 122 321 bp

6, 12, t(9;ll)(q22;q23) AF9(9q22) AF9:1498L22 77 A 1AF9:4092L25 108 208 bp 6 variants type A+B ALLl(llq23) ALLlAF9:4143I-24 48 AF9:1466L26 105 to

24,

368 bp

18A,

18B) t(l;ll)(q21;q23) AFlq(lq21) A ,1AF1Q:421XL22 64 AF1Q:580L20 127 287 bp 3 variants A l(llq23) to

CO 401 bp

CZ CD CO t(ll;19)(q23,-pl3.3) A Ll(llq23) ALL1ENL:4215 22 42 A 1EN :4164 19 112 73 bp 3 variants — f EN (19pl3) to

187 bp m o R6 t(9;22)(q34;qll) ABL(9q34) BCR1AB :1698U19 54 BCR1ABL:1777U19 85 320 bp ela2 types ela2, b2a2,b3a2 BCR(22qll) rπ BCR1AB -.2093 20 83 BCR1ABL:2074 23 84 472 bp b3a2 m (NP: BCR2ABL.3060U23 59 BCR2ABL:3128U22 91 397 bp b2a2

13,

JO z t(9;12)(q34;pl3) ABL(9q34) TELPDGF:309U21 63 TEI-PDGF:343U24 99 366 bp 19 r— TEL(12ρl3) m 20, r 43) t(5;12)(q33,-pl3) PDGFβ(δq33) TELPDGF:834L22 45 TE PDGF:642L22 94 321 bp TEL(12pl3)

R7 t(6;9)(p23;q34) CAN(9q34) DEKCAN:870U24 74 DEKCAN:892U21 89 320 bp

DEK(6p23) DE CAN:1422 21 66 SETCAN:925 20 120

JKP:

?t(9;9) CAN(9q34) SETCAN:468U23 43

14, SETCAN:552tT24 126 393 bp

SET(9q34)

15,

42) t(3;3)(q2Uq2β) Activation of AML1EVI:4331U23 47 AM 1EVL4509U21 101 inv(3#)(q21 q26) EVIl(3q26) AML1EVI:4866L21 75 AML1EVI:4746L25 123 262 bp t(2;3)(p21;q26) t(3;21)(q2β;q22) t(3,5)(q26;q34)

R8 t(ll;17)(q23;q21) P ZF(llq23) P ZFRARA:1092U21 46 P ZFRARA:1252U21 121 315 bp types A+B RARA(17q21) BCR3:1460L19 33 BCR3:1428 22 96 402 bp

(NP: 23, t(15;17)(q21;q22) PML(15q22) BCR1:1338U19 40 BCR1:1497U21 111 427 bp L-form

CO 30, L-, V-, S-forms RARA(17q21) BCR3:988U19 71 BCR3:1057U20 92 ±427 bp V-form

CD 393 bp S-form

31, CO

9, t(2;5)(p23;q35) ALK(2p23) ALKNPM:200U25 35 A KNPM:313U21 117 302 bp

50, NPM(5q35) ALKNPM:627L21 41 A KNPM:590L19 107 rπ 51) co t(5;17)(q35;q22) NPM(5q35) 105 bp S-form c m S-, L-forms RARA(17q21) 234 bp L-form rπ t(3;5)(q25.1;q34) MLF(3q25.1) MLF1:235 27 38 MLF1:192:L28 133 289 + 333 4 splice variants

33 NPM(5q34) 362 + 406 m to 41NP Positive control E2A(19pl3) E2APRL:220U21 78 E2A:1173U19 103 694 bp Included in all R co Ξ2A:1883L22 82 E2A:1844L19 90 and NP mixes

M-mix no. PCR Primers, SEQ ID NO: PCR mix PCR Primers, SEQ ID NO: PCR mix

1st PCR pmol/μl 2nd PCR pmol/μl

ALLAF10A:3730U20 49 5 ALL1AF4:3750U20 139 10

ALL1AF6:4074L21 50 5 ALL1AF6:4037L22 109 10

ALLELL:4236L23 34 5 ALLELL:4191L22 132 10

ALL1:3955U23 143 5 ALL1:3996U24 88 10

AFX:812L20 135 5 AFX-.697L20 140 10

Ml ALL1:351L23 136 5 ALL1:335L22 141 10

ALL1:3181L20 137 5 ALL1:3067L21 142 10

E2A:1045U21 138 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂θ H₂0

ALLAF10A:3730U20 49 5 ALL1AF4:3750U20 139 10

ALL1:3955U24 36 5 ALL1:3995U22 143 10

AL 1 F1:4048L22 65 5 ALL1AF1:3907L27 144 10

AF17:1937L21 51 5 ALL1AF17:4032L22 145 10

M2 ALLAF10A:3968L23 56 5 ALLAF10A:3932L21 134 10

ALLAF10B:4031L22 79 5 ALLAF10B:3997L22 146 10

AF10:728L22 76 5 AF10-.685L21 115 10

E2A:1045U21 138 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2AU844L19 90 7.5

H₂0 H₂θ

E2APRL:696L18 61 5 E2APRL:675L19 150 10

SIL:24U18 147 5 SIL:83U20 98 10

TAL1:203L20 148 5 TAL1U79L20 86 10

M3 E2AHLF:1685L20 62 5 E2AHLP:1543L20 100 10

TELAML1:871U23 44 5 TELAML1:944U23 104 10

TELAML1:1342L23 149 5 TELAML1U168L18 151 10

E2A:1045U21 138 5 E2A:1173U19 103 10

E2A:1883 22 82 5 E2A:1844L19 90 7.5

H₂0 H₂0

AML1EVI:1897U21 60 5 AML1MTG8:1895U20 128 10

AML1EVI:2376L24 152 5 AML1EVI:2345L21 97 10

AML1MTG8:2259L21 153 5 AML1MTG8:2226L22 102 10

TLSERG:649U19 69 5 TLSERG:695U20 156 10

M4 EWSERG:979L22 72 5 TLSERG:945L19 116 10

EAP:990L22 154 5 EAP:781L20 157 10

AMLEVI:2776L22 155 5 AMLEVI:2720L22 158 10

E2A:1045U21 138 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂0 H₂0

ALLAF10A:3730U20 49 5 ALL1AF4:3750U20 139 10

ALL1:3955U24 36 5 ALL1:3996U23 143 10

ALL1AF4:4393L25 159 5 ALL1AF4:4291 25 122 10

ALL1AF9:4143L24 48 5 ALL1AF9:4092L24 108 10

M5 AF9:1498L22 77 5 AF9U466L26 105 10

ALL1AF1Q:4281L20 160 5 AF1Q:580L20 127 10

ALL1ENL:4195L19 161 5 ALL1ENL:4164L19 112 10

ENL:1321L21 162 5 ENL1256L19 165 10

E2A:1045U21 138 5 E2AU173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂0 H₂0

M-mix no. PCR Primers, SEQ ID NO: PCR mix PCR Primers, SEQ ID NO: PCR mix

1st PCR pmol/μl 2nd PCR pmol/μl

BCR1ABL:1698U19 54 5 BCR1ABL:1777U19 85 10

BCR2ABL:3060U23 59 5 BCR2ABL:3128U22 91 10

BCR1ABL:2093L20 83 5 BCR1ABL:2074L23 84 10

46 TEL:56U24 166 5 TEL:114U19 169 10

TELPDGF:595L22 167 5 TELPDGF:555L23 170 10

MN1:5019L25 168 5 MN1:4884L21 171 10

E2A:1045U21 138 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂0 H₂0

DEKC N:667U20 172 5 DEKCAN.-892U21 89 10

DEKCAN:1427L19 173 5 SETCAN:925L20 120 10

SETCAN:468U22 174 5 SETCAN:552U24 126 10

Λ7 CBFBMYHC:269U20 175 5 CBFBMYHC:344U21 129 10

CBFBMYHC:752L22 39 5 CBFBMYHC:595L19 125 10

MYH11:1377L20 176 5 CBFBMYHA:818L21 177 10

E2A:1045U21 138 5 E2AU173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H,θ H,0

PLZFRARA:1092U21 46 5 PLZFRARA:1252U21 121 10

BCR1:1338U19 40 5 BCR1:1497U21 111 10

BCR3:988U19 71 5 BCR3:1057U20 92 10

BCR3U460L19 33 5 BCR3:1428L22 96 10

M8 ALKNPM:200U25 35 5 ALKNPM:313U21 117 10

ALKNPM:627L21 41 5 ALKNPM:590L19 107 10

MLF1:235L27 38 5 MLF1:192L28 133 10

E2A:1045U21 138 5 E2A:1173U19 103 7.5

E2A:1883L22 82 5 E2A:1844L19 90 7.5

H₂0 H_jO Chromosomal alteration included in the R-mix Multiplex RT-PCR analysis.

Chromosomal Genes Fusion gene § NP-mix PCR Mix Size of PCR Positive Presence alteration involved No No. +. Fragment # cell line inv(16Xpl3q22) CBFβ (16q22) CBFβ/MYHll (A) 2 R1A 270 ME-1 * AML

MYHll (16pl3) CBFβ/MYHll (B) 4 RIB 483 AML

CBFβ/MYHll (C) 4 RIB 663 AML

CBFβ/MYHll (D) 2 R1A 337 AML

CBFβ/MYH 11 (E) 2 R1A 544 AML

CBFpVMYHll (F) 4 RIB 174 AML

CBFβ/MYH 11 (G) 4 RIB 241 AML

CBFβ/MYHll (H) 4 RIB 348 AML t(X;liχql3;q23) MLLl (llq23) MLLex6/AFX 46 R1C 448 T

AFX (Xql3) MLLex7/AFX 46 R1C 235 (480) T

MLLex8/AFX 46 R1C 449 (694) Karpas-45» ALL

MLLex9/AFX 46 R1C 596 (841) T t(6;liχq27;q23) MLLl (Uq23) MLLex6/AF6 16 RID 308 ML-2 AMU ALLf

AF6 (6q27) MLLex7/AF6 16 RID 195 (440) AML

MLLex8/AF6 16 RID 309 (594) T

MLLex9/AF6 16 RID 456 (741) T t(ll;19Xq23;pl3.1) MLLl (llq23) MLLex6/ELL 25 R1E 330 T

ELL (19pl3.1) MLLex7 ELL 25 R1E 217 (462) AML

MLLex8/ELL 25 R1E 301 (576) T

MLLex9/ELL 25 R1E 448 (723) T

MLLex6/ELL-insl20 25 R1E 450 T

MLLex7/ELL-insl20 25 R1E 337 (582) AML

MLLex8/ELL-insl20 25 R1E 451 (696) AML

MLLex9/ELL-insl20 25 R1E 598 (845) T

«l;HXp32;q23) MLLl (llq23) MLLex6/AF-lp 5 R2A 300 ALL

AF-lp (lp32) MLLex7/AF-lp 5 R2A 187 (432) T

MLLex8/AF-lp 5 R2A 301 (546) T

MLLex9/AF-lp 5 R2A 448(693) T t(ll;17χq23;q21) MLLl (llq23) MLLex5/AF17 22 R2B 281 AML AF17 (17q21) t(10;liχp!2;q23) MLLl (llq23) MLLex5/AF10 (A:2222) 21A R2C 202 AML

AF10 (10pl2) MLLex6/AF10 (B:979) 21B R2D 270 AML

MLLex7/AF10 (B. 79) 21B R2D 157(402) AML

MLLex8/AF10 (B:979) 21B R2D 271 (516) T

MLLex9/AF10 (B:979) 21B R2D 418 (663) T

MLLex6/AF10 (C:2110) 21A R2C 388 AML

MLLex7/AF10 (C:2110) 21A R2C 275 (520) T

MLLex8/AF10 (C:2110) 21A R2C 389 (634) T

MLLex9/AF10 (C:2110) 21A R2C 536 (781) T

MLLex6/AF10 (D:883) 21B R2D 366 AML

MLLex7/AF10 (D:883) 21B R2D 253 (498) AML

MLLex8/AF10 (D.883) 21B R2D 367 (612) T

MLLex9/AF10 (D:883) 21B R2D 514(759) T

MLLex6/AF10 (E:S89) 21E R2E 267 AML

MLLex7/AF10(E:589) 21E R2E 154(399) T MLLex8/AF10 (E:589) 21E R2E 268(513) T

MLLex9/AF10 (E:589) 21E R2E 415(660) T

MLLex5/AF10(F:1931) 21A R2C 493 AML dupMLL(llq23) MLL(llq23) MLLex5/MLLex2 49 R2F 184 ALL

MLL(llq23) MLLex6/MLLex2 49 R2F 258 AML ALL

MLLex7/MLLex2 49 R2F 145 (390) AML

MLLex8/MLLex2 49 R2F 259 (504) AML

MLLex9/MLLex2 49 R2F 406(651) AML

t(l;19Xq23;pl3) E2A(19pl3) E2A/PBX1 (I) 8 R3A 376 697 ALL

PBX1 (lq23) E2A/PBX1 (la) 8 R3A 403 ALL t(17;19Xq22;pl3) E2A(19pl3) E2Aexl3HLFex4 (I) 33 R3B 390 HAL-01 ALL

HLF (17q22) E2Aexl3insHLFex4 (I) 33 R3B 417 ALL

E2Aexl2/HLFex4 (II) 33 R3B 207 ALL t<12;2iχpl3;q22) TEL(12pl3) TEUAML1 44 R3C 293 ALL

AMLl (21q22) TELAML1 44 R3C 332 ALL

TAL1^D SIL(lp34) SILTAL1 dl+d2 45 R3D 183 RPMI8402 T-ALL TALI (lp34)

t(8;2iχq22;q22) AMLl (21q22) AMLlex5ETO 17 R4A 353 Kasumi-1* AML MGT8 (8q22) t(3;21Xq26;q22)$ AMLl (21q22) AMLlex5MDSl/(EVIl) 11 R4B 446 SKH1* CML-BC, AML, MDSl (3q26) MDS

(EVI1) (3q26) AMLlex6/MDSl/(EVIl) 11 R4B 638 CML-BC, AML, MDS t(16;2iχpll;q22) TLS(16pll) TLS/ERG (a) 32 R4C 318 UTP-L12* AML

ERG (21q22) TLS/ERG(b) 32 R4C 274 UTP-L12» AML

TLS/ERG (c) 32 R4C 239 UTP-L12* AMUALLf

TLSERG (d) 32 R4C 344 ALL

TLS/ERG (e) 32 R4C 413 ALL t(7;10Xq35;q32) Activation of HOXllα 48 R4D 212 RPMI8402 T-ALL, t(10;14Xq24;qll) HOXll (10q32) AML, ALL, CML

t(l;llXq21;q23) MLLl(llq23) MLLlex6/AFlq 6 R5E 300 AMMOL

AFlq(lq21) MLLlex7/AFlq 6 R5E 187 T

MLLlex8/AFlq 6 R5E 301 (546) T

MLLlex9/AFlq 6 R5E 448 (693) T

«4;llXq21;q23) MLLl(llq23) MLLex6/AF4(a:1414) 12 R5A 317 ALL

AF4(4q21) MLLex7/AF4(a:1414) 12 R5A 204 (449) RS4;11 ALL

MLLex8/AF4(a:1414) 12 R5A 318(563) ALL

MLLex9/AF4(a:1414) 12 R5A 465 (710) T

MLLex6/AF4(b:1459) 12 R5A 272 MV4;11 ALL

MLLex7/AF4(b:1459) 12 R5A 159(404) ALL

MLLex8/AF4(b:1459) 12 R5A 273 (518) ALL

MLLex9/AF4(b:1459) 12 R5A 420 (665) T

MLLex6/AF4(c:1546) 12 R5A 185 T

MLLex7/AF4(c:1546) 12 R5A 72(317) ALL

MLLex8/AF4(c:1546) 12 R5A 186(431) ALL MLLex9/AF4 (o 1546) 12 RSA 333 (578) t(l l,19Xq23,pl3 3) MLL (llq23) MLLex6 ENL (A 177) 24 R5B 186 ALL ENL (19pl3 3) MLLex7/ENL (A 177) 24 R5B 73 (318) KOCL-44* ALL MLLex8/ENL (A 177) 24 R5B 187 (432) KOCL-44* ALL MLLex9 ENL (A 177) 24 R5B 334 (579) T t(9,l l)(q22,q23) MLL (llq23) MLLex6/AF9 (A) 18A R5C 321 AML AF9 (9q22) MLLex7/AF9 (A) 18A R5C 208 (453) Mono-Mac-6 AML MLLex8/AF9 (A) 18A R5C 322 (567) Mono-Mac-6 AML MLLex9/AF9 (A) 18A R5C 469 (714) T MLLex6/AF9 (B) 18B R5D 367 AML MLLex7/AF9 (B) 18B R5D 254 (499) T MLLex8/AF9 (B) 18B R5D 368 (613) T MLLex9/AF9 (B) 18B R5D 515 (760) T

t(9,22Xq34,qll) BCR(22ql l) BCR/ABL ela2 19 R6A 320 ALL

ABL (9q34) BCR ABL b2a2 20 R6B 397 CML

BCR/ABL b3a2 20 R6B 472 CML t(9,12)(q34,pl3) TEL (12pl3) TEL/ABL 43 R6C 366 ALL ABL (9q34) t(5,12)(q33,pl3) TEL (12pl3) TEL/PDGFRβ 13 R6D 321 CMML, MDS PDGFRβ (5q33)

t(6,9)(q23,q34) DEK (6q23) DEK CAN 14 R7A 320 AML CAN (9q34)

^■*(9,9) SET (9q34) SET/CAN 15 R7B 393 AUL CAN (9q34) t(3,3Xq21,q26) Activation of EVIlα 42 R7C 262 JOSK-I AML ιnv(3)(q21q26) EVI1 42 R7C 262 CML-BC, AML ιns(3Xq21q25q26) 42 R7C 262 AML t(3,4)(q26,q21) 42 R7C 262 MDS, AML t(3,12,20)(q26,ql2,ql3) 42 R7C 262 CML-BC t(3,21)(q26,q22)$ AMLl (21q22) AM lex5/(MDSl)/EVU 42 R7C 262 SKH1* CML-BC, AML,

EVI1 (3q26) AMLlex5/EVIl 42 R7C 262 MDS t(ll;17Xq23;q21) PLZF (llq23) PLZF/RARα(A:1365) 23 R8A 315 APL

RARAα(17q21) PLZF/RARα (B:1452) 23 R8A 402 APL t(15;17Xq21;q22) PML(15q21) PMLex3/RARαex2 30 R8C 393 NB4 APL RARα(17q21) S-form (=BCR3)

PMLex3Δ/RARαex2 30 R8C 338 NB4 APL S-form splice variant

PMLex6/RARαex2 31 R8B 427 NB4 APL L-form (=BCR1)

PMLex3Δex5+6/ 30 R8C 464 NB4 APL

RARαex2,

L-form splice variant

PMLΔex6-(+/-)ins- 31 R8B +/-427 APL RARαex2 V-form (=BCR2) CL, T-/ B-cell

K2;5χp23;q35) NPM (5q35) NPM/ALK 9 R8D 302 Karpas-299 AL lymphomas ALK (2p23) t(5;17Xq35;q22) NPM (5q35) NPM(S)/RARα 51 R8E 105 APL RARα(17q21) NPM(LyRARα 51 R8E 234 APL ML t(3;5Xq25.1;q34) NPM(5q35) NPM/MLF1 50 R8F 289 MDS, A MLFl (3q25.1)

Abbreviations: No, number; t, translocation; inv, inversion; p, short chromosome arm; q, long chromosome arm; ex, exon; ins, insertion; T, theoretically possible translocation variant; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; CML (-BC), chronic myeloid leukemia (in blast crisis); MDS, myelodysplastic syndrome; APL, acute promyelocytic leukemia; AUL, acute undifferenti- ated leukemia; CMML, chronic myelomonocytic leukemia; ALCL, anaplastic large cell lymphoma; AMMOL, acute myelomonocytic leukemia.

§ Letters and numbers in parentheses after the fusion-gene indicates alternative breakpoints and/or splice variants, t R1-R8, indicate multiplex reaction number, suffix A to F indicate split-out reaction.

* Cell line not available for testing described as positive: ME-1„ Karpas-45, Kasumi-1, SKHl, UTP-L12, KOCL-44.

# Numbers in parentheses indicates the size of the co-amplified PCR fragment resulting from the MLLl exon 5 primer. π No fusion mRNA is generated, only expression of the gene is tested for.

$ The t(3;21)(q26;q22) resulting in a AMLl/MDS/EVIl fusion will be detected in multiplex reaction R4 and R7. Chromosomal alteration included in the M -mix Multiplex RT-PCR analysis.

Chromosomal Genes Fusion gene § M-NP PCR Mix Size of PCR Positive Presence alteration involved mix No No. J Fragment # cell line dupMLL (llq23) MLL (l lq23) MLLex5/MLLex2 M49 MIA 186 ALL

MLL (llq23) MLLex6/MLLex2 M49 MIA 260 AML, ALL

MLLex7/MLLex2 M49 MIA 147 (392) AML

MLLex8/MLLex2 M49 MIA 261 (506) AML

MLLex9/MLLex2 M49 MIA 408 (653) AML dupMLL (llq23) MLL (l lq23) MLLex5/MLLex4 M101 M1B 193 T

MLL (llq23) MLLex6/MLLex4 M101 M1B 267 AML

MLLex7/MLLex4 M101 M1B 154 (399) T

MLLex8/MLLex4 MlOi M1B 268 (513) T

MLLex9 MLLex4 M101 M1B 415 (660) T t(X;l l)(ql3;q23) MLLl (l lq23) MLLex6/AFX M46 MIC 245 T

AFX (Xql3) MLLex7/AFX M46 MIC 132 (377) T

MLLex8/AFX M46 MIC 246 (491) Karpas-45* ALL

MLLex9/AFX M46 MIC 393 (638) T t(6;liχq27;q23) MLLl (llq23) MLLex6/AF6 M16 MID 309 ML-2 AML, ALLt

AF6 (6q27) MLLex7/AF6 M16 MID 195 (441) AML

MLLex8/AF6 M16 MID 309 (595) T

MLLex9/AF6 M16 MID 456 (742) T t(ll;19)(q23;pl3.1) MLLl (llq23) MLLex6/ELL M25 M1E 331 T

ELL (19pl3.1) MLLex7/ELL M25 M1E 217 (463) AML

MLLex8/ELL M25 M1E 301 (577) T

MLLex9/ELL M25 M1E 448 (724) T

MLLex6/ELL-insl20 M25 M1E 451 T

MLLex7/ELL-insl20 M25 M1E 337 (583) AML

MLLex8/ELL-insl20 M25 M1E 451 (697) AML

MLLex9/ELL-insl20 M25 M1E 598 (846) T

t(l;liχp32;q23) MLLl (llq23) MLLex6/AF-lp M5 M2A 183 ALL

AF-lp (lp32) MLLex7/AF-lp M5 M2A 70 (315) T

MLLex8/AF-lp M5 M2A 184 (429) T

MLLex9/AF-lp M5 M2A 331 (576) T t(ll;17χq23;q21) MLLl (llq23) MLLex5/AF17 M22 M2B 282 AML AF17 (17q21) t(10;l l)(pl2;q23) MLLl (Ilq23) MLLex5/AF10 (A:2222) M21A M2C 203 AML

AF10 (10pl2) MLLex6/AF10 (B:979) M21B M2D 271 AML

MLLex7/AF10 (B:979) M21B M2D 158 (403) AML

MLLex8/AF10 (B:979) M21B M2D 272 (517) T

MLLex9/AF10 (B:979) M21B M2D 419 (664) T

MLLex6/AF10 (C:2110) M21A M2C 389 AML

MLLex7/AF10 (C:2110) M21A M2C 276 (521) T

MLLex8/AF10 (C:2110) M21A M2C 390 (635) T

MLLex9/AF10 (C:2110) M21A M2C 537 (782) T

MLLex6/AF10 (D:883) M21B M2D 367 AML

MLLex7/AF10 (D:883) M21B M2D 254 (499) AML

MLLex8/AF10 (D:883) M21B M2D 368 (613) T MLLex9/AF10(D:883) M21B M2D 515(760) T

MLLex6/AF10 (E:589) M21E M2E 268 AML

MLLex7/AF10 (E:589) M21E M2E 155(400) T

MLLex8/AF10 (E:589) M21E M2E 269(514) T

MLLex9/AF10 (E:589) M21E M2E 415 (661) T

MLLex5/AF10(F:1931) M21A M2C 494 AML

t(l;19Xq23;pl3) E2A(19pl3) E2A/PBX1 (I) M8 M3A 376 697 ALL

PBX1 (lq23) E2A/PBX1 (la) M8 M3A 403 ALL t(17;19Xq22;pl3) E2A(19pl3) E2Aexl3/HLFex4 (I) M33 M3B 390 HAL-01 ALL

HLF (17q22) E2Aexl3insHLFex4 (I) M33 M3B 417 ALL

E2Aexl2/HLFex4 (II) M33 M3B 207 ALL t(12;2iχpl3;q22) TEL(12pl3) TELAML1 M44 M3C 242 ALL

AMLl (21q22) TEL/AML1 M44 M3C 281 ALL

TAL1^D SIL(lp34) SILTAL1 dl+d2 M 5 M3D 183 RPMI8402 T-ALL TALI (lp34)

t(8;2iχq22;q22) AMLl (21q22) AMLlexS/ETO M17 M4A 353 Kasumi-1* AML MGT8 (8q22) t(3;2iχq26;q22) AMLl (21q22) AMLlex5/MDSl/(EVU) Mil M4B 446 SKHl* CML-BC, AML, MDSl (3q26) MDS

(EVU)(3q26) AMLlex6/MDSl/(EVIl) Mil M4B 638 CML-BC, AML, MDS t(3;21Xq26;q22) AMLl (21q22) AMLlex5/EVIlex2 M102 M4C 540 AML EVI1 (3q26) AMLlex5EVllex3 M102 M4C 350 AML t(3;2iχq26;q22) AMLl (21q22) AMLlex5/EAP M103 M4D 475 AML EAP (3q26) AMLlex6/EAP M103 M4D 355 AML t(16;2iχpll;q22) TLS(16pll) TLS/ERG (a) M32 M4E 313 UTP-L12» AML

ERG (21q22) TLS/ERG (b) M32 M4E 269 UTP-L12* AML

TLS/ERG (c) M32 M4E 234 UTP-L12* AML, ALL

TLSERG (d) M32 M4E 339 ALL

TLS/ERG (e) M32 M4E 408 ALL

t(4;liχq21;q23) MLLl (llq23) MLLex6/AF4 (a:1414) M12 M5A 318 ALL

AF4(4q21) MLLex7/AF4 (a:1414) M12 M5A 204(450) RS4;11 ALL

MLLex8/AF4(a:1414) M12 M5A 318(564) ALL

MLLex9/AF4(a:1414) M12 M5A 465(711) T

MLLex6/AF4(b:1459) M12 M5A 273 MV4;11 ALL

MLLex7/AF4(b:1459) M12 M5A 159(405) ALL

MLLex8/AF4(b:1459) M12 M5A 273(519) ALL

MLLex9/AF4(b:1459) M12 M5A 420(666) T

MLLex6/AF4(o:1546) M12 M5A 186 T

MLLex7/AF4(c:1546) M12 M5A 72(318) ALL

MLLex8/AF4(c:1546) M12 M5A 186(432) ALL

MLLex9/AF4(c:1546) M12 M5A 333(579) T t(ll;19Xq23;pl3.3) MLL(llq23) MLLex6ENL(A.177) M24 M5B 187 ALL ENL(19pl3.3) MLLex7/ENL(A177) M24 M5B 73(319) KOCL-44* ALL MLLex8ENL(A:177) M24 MSB 187(433) KOCL-44* ALL MLLβx9ENL(A177) M24 MSB 334(580) T t(ll;19Xq23;pl3.3) MLL(llq23) MLLex6ENL (B:) M24 M5C 315 KOPN-1* ALL

ENL(19pl3.3) MLLex7/ENL (B:) M24 M5C 201 (447) T

MLLex8ENL(B:) M24 MSC 316(561) T

MLLex9ENL(B:) M24 MSC 463(708) T t(9;liχq22;q23) MLL(llq23) MLLex6/AF9(A) M18A MSD 322 AML AF9 (9q22) MLLex7/AF9(A) M18A M5D 208 (454) Mono-Mac-6 AML MLLβx8/AF9 (A) M18A MSD 322 (568) Mono-Mac-6 AML MLLex9/AF9(A) M18A M5D 469 (715) T MLLex6/AF9(B) M18B MSE 368 AML MLLex7/AF9 (B) M18B M5E 254 (500) T MLLex8/AF9 (B) M18B M5E 368 (614) T MLLex9/AF9(B) M18B MSE 515 (761) T t(l;πχq21;q23) MLLl (Uq23) MLLlex6/AFlq M6 MSF 301 AMMOL

AFlq(lq21) MLLlex7/AFlq M6 M5F 187(426) T

MLLlex8AFlq M6 MSF 301 (547) T

MLLlex9/AFlq M6 MSF 448 (694) T

«(9;22Xq34;qll) BCR(22qll) BCR/ABL ela2 19 M6A 320 ALL

ABL(9q34) BCR/ABL b2a2 20 M6B 397 CML

BCR/ABL b3a2 20 M6B 472 CML t(9;12Xq34;pl3) TEL(12pl3) TELABL M43 M6C 595 ALL ABL(9q34) t(5;12Xq33;pl3) TEL(12pl3) TEL/PDOFRβ M13 M6D 472 CMML.MDS PDGFRβ (5q33) t(12;22χpl3;qll) TEL(12pl3) TEL/MN1 M10S M6E 244 AML MNl(22qll) M105 M6E 409 AML

t(6;9Xq23;q34) DEK(6q23) DEKCAN M14 M7A 320 AML CAN(9q34)

?t(9;9) SET(9q34) SET/CAN M15 M7B 393 AUL CAN (9q34) inv(16Xpl3q22) CBFβ(16q22) CBFpVMYHll(A) M2 M7C 270 ME-1* AML MYHll(16pl3) CBFβMYHll(B) M4 M7D 434 AML CBFp7MYHll(C) M4 M7D 614 AML CBFpVMYHll(D) M2 M7C 337 AML CBFpVMYHll(E) M2 M7C S44 AML CBFB/MYH11(F) M4 M7D 125 AML CBF7MYHU(G) M4 M7D 192 AML CBFpVMYHll(H) M4 M7D 299 AML t(l l;17Xq23;q21) PLZF (l lq23) PLZF/RARα(A1365) 23 M8A 315 APL RARAα (17q21) PLZF/RARα(B:1452) 23 M8A 402 APL t(15;17Xq21;q22) PML(15q21) PMLex3/RARαex2 30 M8C 393 NB4 APL RARα(17q21) S-form (=BCR3)

PMLex3Δ/RARαex2 30 M8C 338 NB4 APL S-form splice variant

PMLex6/RARαex2 31 M8B 427 NB4 APL L-form (=BCR1)

PMLex3Δex5+6/ 30 M8C 464 NB4 APL

RARαex2,

L-form splice variant

PMLΔex6-(+/-)ins- 31 M8B +/-427 APL

RARαex2

V-form (=BCR2) t(2,5)(p23;q35) NPM (5q35) NPM ALK 9 M8D 302 Karpas- 299 ALCL, T-/ B-cell ALK (2p23) lymphomas

t(5;17Xq35;q22) NPM (5q35) NPM(S)/RARα 51 M8E 105 APL

RARα(17q21) NPM(L)/RARα 51 M8E 234 APL

t 3;5Xq25.1;q34) NPM (5q35) NPM/MLF1 50 M8F 289 MDS, AML MLF1 (3q25.1)

§ Letters and numbers in parentheses after the fusion-gene indicates alternative breakpoints and or splice variants. J R1-R8, indicate multiplex reaction number, suffix A to F indicate split-out reaction.

* Cell line not available for testing described as positive: ME-1„ Karpas-45, Kasumi-1, SKHl, UTP-L12, KOCL-44, KOPN-1.

# Numbers in parentheses indicates the size of the co-amplified PCR fragment resulting from the MLLl exon 5 primer.

Nested RT-PCR Primers

M-NP-mix ver.1

M-NP Rearrangement Genes rt-Primers Nl SEQ ID PCR-Primers N2 SEQ ID Pos. Controle mix (1st PCR) No: (2nd PCR) No:. no.

CO CBF/8 (16q22) CBFBMYHC:269U20 175 CBFBMYH:344U21 129 Pt:

CD CO M2 inv(16)(pl3;q22) MYHl l(16pl3) CBFBMYHC:752L22 39 CBFBMYHC:595L19 125

M4 inv(16)(pl3;q22) CBF0 (16q22) CBFBMYHC:269U20 175 CBFBMYH:344U21 129 No m MYHl l(16pl3) MYH11: 1377L20 176 CBFBMYHA:818L21 177 m M45 tall^d1-³ SILl(lp34) SIL:24U18 147 SIL:83U20 98 RPMI

(40 kb deletion) TALl(lp34) TAL1:203L20 148 TAL1:179L20 86

33

M49 dup(l lq23) ALLl(l lq23) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 Pt m r dup exon 2-5/9 ALL13955U23 36 ALL1:3996U24 88

ALL1:351L23 136 ALL1:335L22 141

M101 dup(l lq23) ALLl(l lq23) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 No dup exon 4-5/9 ALL13955U23 36 ALL1:3996U24 88

ALL1:3181L20 137 ALL1:3067L21 142

AFXl(X;ql3) ALLAF10A:3730U20 49 ALL1AF4:3751U20 139 Cell line:

M46 t(X;l l)(ql3;q23) ALLl(l lq23) ALL1:3955U24 36 ALL1:3996U24 88 Karpas 45

AFX:812L20 135 AFX:697L20 140

AFlp(lp32) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 No

M5 t(l l l)(p32;q23) ALLl(l lq23) ALL1:3955U24 36 ALL1:3995U22 143

ALL1AF1:4048L22 65 ALL1AF1:3907L27 144

M-NP Rearrangement Genes PCR-Primers Nl SEQ ID P<_R-Primers N2 SEQ ID Pos. Controle mix (1st PCR) No: (2nd PCR) No:. no.

AFlq(lq21) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 No t(l;ll)(q21;q23) ALLl(llq23) ALL1:3955U24 36 ALL1:3996U23 164

M6 ALL1AF1Q:4281L20 160 AF1Q:580L20 127

M8 t(l;19)(q23;pl3) PBXl(lq23) E2A:1045U21 138 E2A:1173U19 103 Pt+Cell line: E2A(19pl3) E2APRL:696L18 61 E2APRL:675L19 150 697

9 t(2;5)(p23;q35) ALK(2p23) ALKNPM:200U25 35 ALKNPM:313U21 117 Pt+ NPM(5q35) ALKNPM:627L21 41 ALKNPM:590L19 107 Karpas 299 co c.; 50 t(3;5)(q25.1;q34) MLF(3q25.1) ALKNPM:200U25 35 ALKNPM:313U21 117 No σ co NPM(5q34) MLF1:235L27 38 MLF1:192:L28 133

Mil t(3;21)(q26;q22) MDS(3q26) AMLIEVI: 1897U21 60 AML1EVI:2345L21 128 Cell line: m AMLl(21q22) AML1EVI:2376L24 97 SKffl o m M102 t(3;21)(q26;q22) EVI-l(3q26) AMLIEVI: 1897U21 60 AML1MGT8:1895U20 128 m AMLl(21q22) AML1EVI:2776L22 155 AML1EVI:2720L22 158

M103 t(3;21)(q26;q22) EAP(3q26) AMLIEVI: 1897U21 60 AML1MGT8:1895U20 128 rπ AMLl(21q22) EAP:990L22 154 EAP:781L20 157 r

M12 t(4;ll)(q21;q23) AF4(4q21) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 Cell line: ALLl(llq23) ALL1:3955U24 36 ALL1:3996U23 164 RS4;11

ALL1AF4:4393L25 159 ALL1AF4:4291L25 122 MV4;11

M13 t(5;12)(q33;pl3) PDGFS(5q33) TEL:56U24 166 TEL:114U19 169 TEL(12pl3) TELPDGF:595L22 167 TELPDGF:555L23 170

51 t(5;17)(q35;q22) NPM(5q35) NPMALK:200U25 35 NPMALK:313U21 117 S-, L-foπns RARA(17q21) BCR3:1460L19 33 BCR3.1428L22 96

M-NP Rearrangement Genes PCR-Primers Nl SEQ ID PCR-Primers N2 SEQ ID Pos. Controle mix (1st PCR) No: (2nd PCR) No:. no.

M14 t(6;9)(p23;q34) CAN(6p23) DEKCAN:667U20 172 DEKCAN:892U21 89 Pt: DEK(9q34) DEKCAN:1427L19 173 SETCAN:925L20 120

M15 ?t(9;9) CAN(6p23) SETCAN:468U22 174 SETCAN:552U24 126 No SET(6p23) DEKCAN:1427L19 173 SETCAN:925L20 120

M16 t(6;ll)(q27;q23) AF6(6q27) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 Pt.+ Cell line: ALL(llq23) ALL1:3955U24 36 ALL1:3996U24 88 ML-2 co c: ALL1AF6:4074L21 50 ALL1AF6:4037122 109

CD O

M17 t(8;21)(q22;q22) MTG8(8q22) AMLIEVI: 1897U21 60 AML1MGT8:1895U20 128 Pt+Cell line: AMLl(21q22) AML1MGT8:2259- 153 AML1MGT8:2226L22 102 Kasumi-1 m L21 co m rπ M18A t(9;ll)(q22;q23) AF9(9q22) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 Cell line: m ALLl(llq23) ALL1:3955U24 36 ALL1:3996U23 164 Mono Mac 6

AF9:1498L22 77 AF9:1466L26 105 m M18B t(9;ll)(q22;q23) AF9(9q22) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 Cell line: r ALLl(llq23) ALL1:3955U24 36 ALL1:3996U23 143 Mono Mac 6

ALL1AF9:4143L24 48 ALL1AF9:4092L25 108

M43 t(9;12)(q34;_P13) ABL(9q34) TEL:56U24 166 TEL:114U19 169 No TEL(12pl3) BCR1ABL:2093L20 83 BCR1ABL:2074L23 84

19 t(9;22)(q34;qll) ABL(9q34) BCR1ABL:1698U19 54 BCR1ABL:1777U19 85 Pt: type ela2 BCR(22qll) BCR1ABL:2093L20 83 BCR1ABL:2074L23 84

20 t(9;22)(q34;qll) ABL(9q34) BCR2ABL:3060U23 59 BCR2ABL:3128U22 91 Pt: type b2a2+b3a2 BCR(22qll) BCR1ABL:2093L20 83 BCR1ABL:2074L23 84 Pt:

M21A t(10;ll)(pl4;q23) AF10(10pl4) ALLAF10:3730U20 49 ALLAF4:3750U20 139 Pt. A+C type ALLl(l lq23) ALL1:3955U24 36 ALL1:3995U22 143

ALLAF10A:3968L23 56 ALLAF10A:3932L21 134

M21B t(10;ll)(pl4;q23) AF10(10pl4) ALLAF10:3730U20 49 ALLAF4:3750U20 139 Pt. B+D type ALLl(llq23) ALL1:3955U24 36 ALL1:3995U22 143 co ALLAF10B:4031L22 79 ALLAF10B:3997L22 146 o M21E t(10;l l)(pl4;q23) AF10(10pl4) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 No E type ALLl(llq23) ALL1:3955U24 36 ALL1:3995U22 143 rπ AF10:728L22 76 AF10:685L21 115

CO

IT M22 t(ll;17)(q23;q21) ALLl(llq23) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 No m A AF-17(17q21) ALL1:3955U24 36 ALL1:3995U22 143

30 AF17:1937L22 51 ALL1AF17:4032L22 145 m 23 t(l l;17)(q23;q21) PLZF(llq23) PLZFRARA:1092U21 46 PLZFRARA:1252U21 121 No t <3o5 B RARA(17q21) BCR3:1460L20 33 BCR3:1428L22 96

M24 t(ll;19)(q23;pl3.3) ALLl(llq23) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 Pt.. A ENL(19pl3) ALL1:3955U24 36 ALL1:3996U23 164 (HB1119+

ALL1ENL:4195L19 161 ALL1ENL:4164L19 112 KOCL33 ect.)

M104 t(l l;19)(q23;pl3.3) ALLl(llq23) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 B ENL(19pl3) ALL1:3955U24 36 ALL1:3996U23 164

ENL:1321L21 162 ENL:1256L19 165

M25 t(ll;19)(q23;pl3.1) ALLl(llq23) ALLAF10A:3730U20 49 ALL1AF4:3750U20 139 Pt. ELL(19pl3) ALL1:3955U24 36 ALL1:3996U24 88

ALLELL:4236L23 34 ALLELL:4191L22 132

M44 t(12;21)(pl3;q22) TEL(12pl3) TELAML1:871U23 44 TELAML1:944U23 104 Pt.

CO AMLl(21q22) TELAML1:1342L23 149 TELAML1:1168L18 151 c CD: O M105 t(12;22)(pl3;qll) TEL(12pl3) TEL:56U24 166 TEL:114U19 169 MNl(22;qll) MN1:5019L25 168 MN14884L21 cr m —i co 30 t(15;17)(q21;q22) PML(15q22) BCR1:1338U19 40 BCR1:1497U21 111171 Pt+Cell line: : mr type V+L RARA(17q21) BCR3:1460L19 33 BCR3:1428L22 96 NB4 m

31 t(15;17)(q21;q22) PML(15q22) BCR3:988U19 71 BCR3:1057U20 92 Pt:

30 type S RARA(17q21) BCR3:1460L19 33 BCR3:1428L22 96 m r M32 t(16;21)(pll;q22) FUS(16pl l) TLSERG:649U19 69 TLS:695U20 156 Pt + ERG(21q22) EWSERG:979L22 72 TLSERG:945L19 116 (UTP-L12)

M33 t(17;19)(q22;pl3) HLF(17q22) E2A:1045U21 138 E2A:1173U19 103 Cell line: E2A(19pl3) E2AHLF:1685L20 62 E2AHLF:1543L20 100 HAL-01

M41 Positive controle E2A(19pl3) E2A:1045U21 138 E2A:1173U19 103 All Pts. and E2A:1883L22 82 E2A:1844L19 90 cell lines

In the following is given the precise details concerning the protocols used for cDNA production and PCR amplification reactions:

' cDNA syntesis (in PCR-Lab #1) :

1. Mix 1 μg totale RNA resuspended in 10 μl DEP H₂0 with 2.5 μl 1 pmol/μl (of each) specific cDNA primer.

2. Incubate 5 in at 65°C, place on ice.

3. Add 12.5 μl McDNA-mix, mix, spin briefly. McDNA- mix (12.5 μl) :

5 μl 5X first strand buffer, 2.5 μl 100 mM DTT,

2.5 μl 10 mM (each) dNTP (Pharmacia), 2.0 μl MoMLV RT 200 u/μl (BRL) , and 0.5 μl 25 u/μl RNase inhibitor (Boehringer) .

4. Incubate 45 min at 37°C.

5. Dilute cDNA to 55 μl with ddH₂0.

1. PCR amplification (in PCR-Lab #1) :

1. To eight 200 μl PCR tubes on ice, add 20 μl of R1A-R8A PCR-mix.

8x RnA PCR-mix made on ice (20 μl each) : 2.5 μl 10X Taq buffer, 0.5 μl 10 mM (each) dNTP,

1 μl primer-mix (either R1A to R8A primer mix) , 15.7 μl ddH₂0, and

0.3 μl 5u/μl AmpliTaq Gold (Perkin Elmer).

2. Add 5 μl diluted cDNA to each of the 8 R1A-R8A mixtures using a Biohit Proline electric dispenser or equivalent. 3. Transfer tubes to a 9600 termocycler (Perkin

Elmer) and run the RA PCR: 95°C 15 min. followed by 25 cycles: 95°C for 30 sec. 58°C for 30 sec. 72°C for 90 sec.^"

2. PCR amplification (in PCR-Lab #2):

1. Withdraw 1 μl from each of the 8 R1A-R8A PCR reactions using a 8 chanel Biohit 0.2-10 μl elec- trie pipette and add it to 8 tubes each with 24 μl R1B-R8B PCR mix.

8x RnB PCR-mix made on ice (24 μl each) : 2.5 μl 10X Taq buffer, 0.5 μl 10 mM (each) dNTP, 1 μl primer-mix (R1B-R8B) ,

19.7 μl ddH₂0, and 0.3 μl 5u/μl AmpliTaq Gold (Perkin Elmer).

2. Transfer tubes to termocycler and start RB program: 95°C 15 min. followed by 20 cycles:

95°C for 30 sec. 58°C for 30 sec. 72°C for 90 sec. , followed by 10 min at 72 °C.

D. Agarose gel electrophoresis (in PCR Lab #3):

1. Withdraw 12.5 μl PCR product, add 4 μl 5X loading buffer and run 15 μl on a 1.5% agarose gel with 25 μl/liter 10 mg/ml EtBr for 60 min at 100 V.

Exemplary results of the inventive methods appear from Figs. 1-3. When a patient serum is negative for the chromosomal abnormalities which can be detected with a given mixture of primers, the only band visible in the gelelectrophoresis is the internal positive control (cf. e.g. lanes 1-3 and 5-8 in Fig. 1) . A positive sample will manifest itself as a band deviating from the position of the internal standard (cf. lane 4 in Fig. 1) . The precise location in the gel can then identify precisely the kind of rearrangement (in this case t(8;21) which results in the positive reaction. Absence of the internal positive control indicates that the method should be repeated since false negatives might be present.

Figs. 2A and B shows essentially the same picture as Fig. 1, although for different chromosomal rearrangements; the gene identified is indicated over each panel.

In cases where it is impossible to determine the precise nature of the genetic rearrangement in a positive sample, the sample is subjected to individual (non-multiplex) PCR reactions using specific primers selected from the NP mixes Listed above. Thereby, the precise variant of the chromosomal rearrangement can be determined (cf. Fig. 4).

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: PALLISGAARD, Niels

(B) STREET: Fasanvej 28

(C) CITY: Aarhus V.

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP) : 8210

(ii) TITLE OF INλ/ENTION: Improved detection of fusion genes (iii) NUMBER OF SEQUENCES: 147

(iv) COMPUTER READABLE FORM:

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(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

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(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: GAGCTGGATG TTGAGAGTGG AGAT 24

(2) INFORMATION FOR SEQ ID NO: 59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: GAGTCACTGC TGCTGCTTAT GTC 23

(2) INFORMATION FOR SEQ ID NO: 60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: GATGGCACTC TGGTCACTGT G 21

(2) INFORMATION FOR SEQ ID NO : 61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: GCCACGCCTT CCGCTAAC 18

(2) INFORMATION FOR SEQ ID NO: 62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: GCCCAGCTCC TTCCTCAAGT 20

(2) INFORMATION FOR SEQ ID NO: 63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: GCTGCTGACC AAAGAGGACT T 21

(2) INFORMATION FOR SEQ ID NO: 64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: GCTTGAGAGG GAAGACAATG AG 22

(2) INFORMATION FOR SEQ ID NO: 65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: GGATACCTTT GCCATCTGTG TC 22

(2) INFORMATION FOR SEQ ID NO : 66:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: GGCAAGGATT TGGTGTGAGA T 21

(2) INFORMATION FOR SEQ ID NO : 67:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: GGGCGTCAAC AACCTCACTG 20 (2) INFORMATION FOR SEQ ID NO: 68: ^"

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: GGGTGACTGG CAGCACAGAT 20

(2) INFORMATION FOR SEQ ID NO: 69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: GGTGGCGGTT ATGGCAATC 19

(2) INFORMATION FOR SEQ ID NO : 70:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:

GTCTGCCGTC TCCACTTTGT C 21

(2) INFORMATION FOR SEQ ID NO : 71:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: GTGCGCCAGG TGGTAGCTC 19

(2) INFORMATION FOR SEQ ID NO: 72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: GTTCATGTTG GGTTTGCTCT TC 22

(2) INFORMATION FOR SEQ ID NO: 73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: TCTCCTATCT CGGGTGAAAT GTC 23

(2) INFORMATION FOR SEQ ID NO: 74:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: TGCCAATGTT AAGAAAGCAG ATAG 24

(2) INFORMATION FOR SEQ ID NO: 75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: TGCCGTGTTA GGTTTGCAGA C 21

(2) INFORMATION FOR SEQ ID NO: 76:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: TGGACATTAT CGGCACCATT AC 22

(2) INFORMATION FOR SEQ ID NO: 77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: TTCGGCTGCC TCCTCTATTT AC 22

(2) INFORMATION FOR SEQ ID NO : 78:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: TTCTCGTCCA GCCCTTCTAC C 21

(2) INFORMATION FOR SEQ ID NO: 79:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: TTGCCCTCTG ACCCTCTAGT CT 22

(2) INFORMATION FOR SEQ ID NO: 80:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 80: TTTAGAGGGG AAAACACAGA TGG 23 (2) INFORMATION FOR SEQ ID NO: 81: ^"

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) ^»

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: TTTGAAGGCT CCCATGATTC TG 22

(2) INFORMATION FOR SEQ ID NO: 82:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: TTTTCCTCTT CTCGCCGTTT CA 22

(2) INFORMATION FOR SEQ ID NO : 83:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:

TTTTGGTTTG GGCTTCACAC 20

(2) INFORMATION FOR SEQ ID NO: 84:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: ACACCATTCC CCATTGTGAT TAT 23

(2) INFORMATION FOR SEQ ID NO: 85:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: ACTGCCCGGT TGTCGTGTC 19

(2) INFORMATION FOR SEQ ID NO : 86:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: AGACCGGCCC CTCTGAATAG 20

(2) INFORMATION FOR SEQ ID NO: 87:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: AGCACGGAGC AGAGGAAGTT G 21

(2) INFORMATION FOR SEQ ID NO: 88:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: AGCAGATGGA GTCCACAGGA TCAG 24

(2) INFORMATION FOR SEQ ID NO: 89:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: AGCAGCACCA CCAAGAAGAA T 21

(2) INFORMATION FOR SEQ ID NO : 90:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: AGGTTCCGCT CTCGCACTT 19

(2) INFORMATION FOR SEQ ID NO : 91:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: CACGTTCCTG ATCTCCTCTG AC 22

(2) INFORMATION FOR SEQ ID NO : 92:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92: CAGCGCGACT ACGAGGAGAT 20

(2) INFORMATION FOR SEQ ID NO : 93:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: CAGCGGTGGC TATGGACAG 19 (2) INFORMATION FOR SEQ ID NO: 94:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94: CATGGGGTCC ACGTAGATGT AC 22

(2) INFORMATION FOR SEQ ID NO: 95:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95: CATGTTGTCC AGCCGCATCA G 21

(2) INFORMATION FOR SEQ ID NO : 96:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 96:

CCCATAGTGG TAGCCTGAGG AC 22

(2) INFORMATION FOR SEQ ID NO : 97:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) ^»

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: CCCCAGGCAT ATTTGACTCT C 21

(2) INFORMATION FOR SEQ ID NO : 98:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: CCCGCTCCTA CCCTGCAAAC 20

(2) INFORMATION FOR SEQ ID NO: 99:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99: CCTCATTCAG GTGATGTGCT CTAT 24

(2) INFORMATION FOR SEQ ID NO : 100:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: CGCCTTGCCC AGTACTTGTC 20

(2) INFORMATION FOR SEQ ID NO: 101:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101: CGTCGAATCA AGACCTGCTT C 21

(2) INFORMATION FOR SEQ ID NO: 102:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102: CGTTGTCGGT GTAAATGAAC TG 22

(2) INFORMATION FOR SEQ ID NO: 103:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103: CTACGACGGG GGTCTCCAC 19

(2) INFORMATION FOR SEQ ID NO: 104:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104: CTCATCGGGA AGACCTGGCT TAC 23

(2) INFORMATION FOR SEQ ID NO: 105:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: CTCCATTTCA GAGTCATTGT CGTTAT 26

(2) INFORMATION FOR SEQ ID NO : 106:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106: CTTCCCCTGG ATGGAGAGTA AC 22 (2) INFORMATION FOR SEQ ID NO: 107: ^"

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107: CTTGGGTCGT TGGGCATTC 19

(2) INFORMATION FOR SEQ ID NO: 108:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: GAGCAAAGAT CAAAATCAAA TGTT 24

(2) INFORMATION FOR SEQ ID NO: 109:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:

GAGGACAGCA TTCGCATATC AG 22

(2) INFORMATION FOR SEQ ID NO: 110:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110: GAGGTTTTCG AGGACTAGTT TTAACTGA 28

(2) INFORMATION FOR SEQ ID NO : 111:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111: GCCAGTGTAC GCCTTCTCCA T 21

(2) INFORMATION FOR SEQ ID NO: 112:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112: GCGATGCCCC AGCTCTAAC 19

(2) INFORMATION FOR SEQ ID NO: 113:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

^■ (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: GCGCATCGGT CATTTTGAG 19

(2) INFORMATION FOR SEQ ID NO: 114:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114: GGACCGCCAA GAAAAGAAGT 20

(2) INFORMATION FOR SEQ ID NO: 115:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115: GGCAAACTGA GCGCATGTTA C 21

(2) INFORMATION FOR SEQ ID NO: 116:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: GGTGCCTTCC CAGGTGATG 19

(2) INFORMATION FOR SEQ ID NO : 117:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117: GGTTCAGGGC CAGTGCATAT T 21

(2) INFORMATION FOR SEQ ID NO: 118:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: GGTTTCTTCT TGGGGGCTTT AACT 24

(2) INFORMATION FOR SEQ ID NO: 119:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: GTAGAGCCAG CCAGAGAAAA CAC 23 (2) INFORMATION FOR SEQ ID NO: 120: ^"

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120: GTCTCTCGCT CTGGCACAAG 20

(2) INFORMATION FOR SEQ ID NO : 121:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: GTGGGCATGA AGTCAGAGAG C 21

(2) INFORMATION FOR SEQ ID NO: 122:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:

GTTTTTGGTT TTGGGTTACA GAACT 25

(2) INFORMATION FOR SEQ ID NO: 123:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123: GAACATAGAG GGCACTGACT GTAAG 25

(2) INFORMATION FOR SEQ ID NO: 124:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124: TCCTCGTCCA GCTGGTCTTG 20

(2) INFORMATION FOR SEQ ID NO: 125:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125: TGAGCGCCTG CATGTTGAC 19

(2) INFORMATION FOR SEQ ID NO: 126:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126: TGAGGAACCA GAGAGCTTCT TTAC 24

(2) INFORMATION FOR SEQ ID NO : 127:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127: TGCTGGCAAT GGGAGCTCTC 20

(2) INFORMATION FOR SEQ ID NO: 128:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128: TGGCTGGCAA TGATGAAAAC 20

(2) INFORMATION FOR SEQ ID NO: 129:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129: TGGGCTGTCT GGAGTTTGAT G 21

(2) INFORMATION FOR SEQ ID NO : 130:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130: TGTCGGCTAA ATCCCAAATC T 21

(2) INFORMATION FOR SEQ ID NO: 131:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 131: TTCCACTAGA GGTGTGTGCA GAG 23

(2) INFORMATION FOR SEQ ID NO: 132:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic)"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132: TTCCCCATGA CTGGAGACAT AC 22 (2) INFORMATION FOR SEQ ID NO: 133: ^"

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133: AAAGGGTTCA GAAAAACTTC TTATCATC 28

(2) INFORMATION FOR SEQ ID NO: 134:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "DNA (synthetic) "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134: AACTGCTGTT GCCTGGTTGA T 21

(2) INFORMATION FOR SEQ ID NO: 135:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135: CTGGCAGCAC AGATGGTTTC 20

(2) INFORMATION FOR SEQ ID NO: 136:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136: AATTTCGGTC AGAGCCACTT CTA 23

(2) INFORMATION FOR SEQ ID NO: 137:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 137: ATGGGTGGAG CAAGAGGTTC 20

(2) INFORMATION FOR SEQ ID NO: 138:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138: ATCTACTCCC CGGATCACTC A 21

(2) INFORMATION FOR SEQ ID NO: 139:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139: AGGACCGCCA AGAAAAGAAG 20

(2) INFORMATION FOR SEQ ID NO: 140:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140: AGGGTTCAGC ATCCACCAAG 20

(2) INFORMATION FOR SEQ ID NO : 141: (i) SEQUENCE CHARACTERISTICS:^'

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141: CTTCTAGGTC TCCCACGAGG TT 22

(2) INFORMATION FOR SEQ ID NO: 142:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 142: GGGCATGTCA TCAGGAAACA C 21

(2) INFORMATION FOR SEQ ID NO : 143:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143: CAGCAGATGG AGTCCACAGG AT 22

(2) INFORMATION FOR SEQ ID NO: 144:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144: GTATTTTTCA TATACAGGAT TCCCACT 27

(2) INFORMATION FOR SEQ ID NO: 145:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ^~ ID NO: 145: GTGGGTAGAA GGGAGGCTAA AG 22

(2) INFORMATION FOR SEQ ID NO: 146:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146: TTCCACTAGA GGTGTGTGCA GA 22

(2) INFORMATION FOR SEQ ID NO: 147:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147: CGACCCCAAC GTCCCAGA 18

(2) INFORMATION FOR SEQ ID NO : 148:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148: CGGTCATCCT GGGGCATATT T 21

(2) INFORMATION FOR SEQ ID NO: 149:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149: TCAGCCGAGT AGTTTTCATC ATT 23

(2) INFORMATION FOR SEQ ID NO : 150: (i) SEQUENCE CHARACTERISTICS:^"

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150: CATGTTGTCC AGCCGCATC 19

(2) INFORMATION FOR SEQ ID NO: 151:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 151: GGCCAGCACC TCCACCAT 18

(2) INFORMATION FOR SEQ ID NO: 152:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152: TCGATCTTCC TTTTGGTCCA TATT 24

(2) INFORMATION FOR SEQ ID NO: 153:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153: GCTGTAGGAG AATGGCTCGT G 21

(2) INFORMATION FOR SEQ ID NO: 154:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154: CCTCCTCTTC TTCGTCCTGG TT 22

(2) INFORMATION FOR SEQ ID NO: 155:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155: TACTGCATGG AAACTTTTGG TG 22

(2) INFORMATION FOR SEQ ID NO : 156:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156: GTGGCTATGG ACAGCAGGAC 20

(2) INFORMATION FOR SEQ ID NO: 157:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157: CCCAGCTTTT CCGTTCACTT 20

(2) INFORMATION FOR SEQ ID NO: 158:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158: TCACAGTCTT CGCAGCGATA TT 22

(2) INFORMATION FOR SEQ ID NO: 159: (i) SEQUENCE CHARACTERISTICS:^"

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159: CACTGTCACT GTCCTCACTG TCACT 25

(2) INFORMATION FOR SEQ ID NO : 160:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160: TTCATCAGCA CCACCAACAC 20

(2) INFORMATION FOR SEQ ID NO : 161:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161: CCTCCGTGGT GGGCTTCTT 19

(2) INFORMATION FOR SEQ ID NO: 162:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162: GCTGTTGTCA CTCTCGCTGT C 21

(2) INFORMATION FOR SEQ ID NO: 163:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163: AGGACCGCCA AGAAAAGAAG 20

(2) INFORMATION FOR SEQ ID NO: 164:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164: AGCAGATGGA GTCCACAGGA TCA 23

(2) INFORMATION FOR SEQ ID NO: 165:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165: GCCTCCTCGC CTGACGAAG 19

(2) INFORMATION FOR SEQ ID NO : 166:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 166: AGCAGGAACG AATTTCATAT ACAC 24

(2) INFORMATION FOR SEQ ID NO : 167:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167: CAATCACCTT CCATCGGATC TC 22

(2) INFORMATION FOR SEQ ID NO: 168: (i) SEQUENCE CHARACTERISTICS:^"

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168: AAAAAACTCA TCCACTCAGC AATAG 25

(2) INFORMATION FOR SEQ ID NO : 169:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 169: GACGCCACTT CATGTTCCA 19

(2) INFORMATION FOR SEQ ID NO: 170:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170: GCCAAAGCAT GATGAGGATG ATA 23

(2) INFORMATION FOR SEQ ID NO : 171:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 171: AGCCACGAAT GTCCCAAATC T 21

(2) INFORMATION FOR SEQ ID NO: 172:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 172: GGAATGGCAA GGAAGGCTAA 20

(2) INFORMATION FOR SEQ ID NO: 173:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 173: TTGGGCAAGG ATTTGGTGT 19

(2) INFORMATION FOR SEQ ID NO: 174:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 174: CACCGAAATC AAATGGAAAT CT 22

(2) INFORMATION FOR SEQ ID NO: 175:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 175: TGAAGGCTCC CATGATTCTG 20

(2) INFORMATION FOR SEQ ID NO: 176:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 176: GCTGGTCTTG CAGGCTGTTC 20

(2) INFORMATION FOR SEQ ID NO: 177: (i) SEQUENCE CHARACTERISTICS:^"

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 177: GCAGCTTCGT AGACACGTTG A 21

(2) INFORMATION FOR SEQ ID NO : 178:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 178: GCCTGACACC TT 12

(2) INFORMATION FOR SEQ ID NO: 179:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 179: CTGCCCACAC C 11

(2) INFORMATION FOR SEQ ID NO : 180:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 180: GCCACTAAGC AG 12

(2) INFORMATION FOR SEQ ID NO: 181:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 181: TAATCCTCGT CTT 13

(2) INFORMATION FOR SEQ ID NO: 182:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 182: GTCCTCTTCA ACC 13

Claims

1. A method for detection of the presence or absence of chromosomal abnormalities, each chromosomal abnormality being ^• associated with a condition in a subject and each chromosomal abnormality being defined by at least one characteristic nucleic acid sequence, the method comprising

wherein the multiplex molecular amplification procedure comprises the use of at least 7 mutually distinct primers in one single reaction mixture, each of the at least 7 mutually distinct primers defining an end of at least one characteristic nucleic acid sequence, and wherein at least one of the at least 7 mutually distinct primers defines the first ends of at least two characteristic nucleic acid sequences, said at least two characteristic nucleic acid sequences each being defined in their opposite ends by mutually distinct primers selected from the remainder of the at least 7 mutually distinct primers, whereby the number of amplified characteristic nucleic acid sequences which can be detected upon conclusion of the amplification reaction is at least ^xn+1, wherein n is the number of the at least 7 mutually distinct primers.

2. A method for detection of the presence or absence of chromosomal abnormalities, each chromosomal abnormality being associated with a condition in a subject and each chromosomal abnormality being defined by at least one characteristic ^' nucleic acid sequence, the method comprising

a) obtaining a sample of nucleic acids derived from a subject which may harbour one of said chromosomal abnormalities,

wherein the multiplex molecular amplification reaction comprises

and wherein at least one of the n mutually distinct primers defines first ends of at least two mutually distinct characteristic nucleic acid sequences, said at least two mutually distinct characteristic nucleic acid sequences being defined in their opposite ends by at least two mutually distinct primers selected from the remainder of the n mutually distinct primers, whereby the number of amplified characteristic nucleic acid sequences which can be detected upon conclusion of the amplification procedure is at least ^xn+1.

3. A method according to claim 1 or 2 , wherein n is 7.

4. A method according to claim 1 or 2 , wherein n is 8.

5. A method according to claim 1 or 2 , wherein n is 9.

6. A method according to claim 1 or 2 , wherein n is 10.

7. A method according to claim 1 or 2 , wherein n is 11.

8. A method according to claim 1 or 2 , wherein n is 12.

9. A method according to claim 1 or 2 , wherein n is 13.

10. A method according to claim 1 or 2 , wherein n is 14.

11. A method according to claim 1 or 2 , wherein n is 15.

12. A method according to claim 1 or 2 , wherein n is 16.

13. A method according to claim 1 or 2 , wherein n is 17.

14. A method according to claim 1 or 2 , wherein n is 18.

15. A method according to claim 1 or 2, wherein n is 19.

16. A method according to claim 1 or 2, wherein n is 20.

17. A method according to claim 1 or 2 , wherein n is 21.

18. A method according to claim 1 or 2 , wherein n is 22.

19. A method according to claim 1 or 2 , wherein n is 23.

20. A method according to claim 1 or 2 , wherein n is 24.

21. A method according to claim 1 or 2 , wherein n is 25.

22. A method according to claim 1 or 2 , wherein n is in the range of 30 to 34.

23. A method according to claim 1 or 2 , wherein n is in the range of 35 to 39.

24. A method according to claim 1 or 2 , wherein n is in the range of 40 to 44.

25. A method according to claim 1 or 2 , wherein n is in the range of 45 to 50.

26. A method according to any of the preceding claims, wherein the sample of nucleic acids derived from the subject is in the form of cDNA.

27. A method according to claim 26, wherein the cDNA is obtained by use of specific or non-specific cDNA primers in a molecular amplification procedure wherein the templates in the procedure are in the form of mRNA derived from the subject.

28. A method according to claim 27, wherein the cDNA primers are specific.

29. A method according to claim 28, wherein the number of cDNA primers is at least 20.

30. A method according to claim 28, wherein the number of cDNA primers is at least 50.

31. A method according to claim 28, wherein the number of cDNA primers is at least 100.

32. A method according to claim 28, wherein the number of cDNA primers is at least 150.

33. A method according to claim 28, wherein the number of cDNA primers is at least 200.

34. A method according to any of claims 26-33, wherein the ^■ conditions for obtaining cDNA derived from the subject are compatible with the conditions of the molecular amplification procedure.

35. A method according to any of the preceding claims, wherein said multiplex molecular amplification is a multiplex polymerase chain reaction.

36. A method according to claim 8, wherein said multiplex polymerase chain reaction is a nested polymerase chain reaction.

37. A method according to any of the preceding claims, wherein the chromosomal abnormality is the presence of a tran- scribed fusion gene.

38. A method according to claim 29, wherein the presence of the transcribed fusion gene is the result of an inversion.

39. A method according to claim 29, wherein the presence of the transcribed fusion gene is the result of a deletion.

40. A method according to claim 29, wherein the presence of the transcribed fusion gene is the result of a duplication.

41. A method according to claim 29, wherein the presence of the transcribed fusion gene is the result of activation of a proto-oncogene, such as Hox-11 and evi-1.

42. A method according to any of the preceding claims, wherein at least one chromosomal abnormality is associated with a malignant neoplastic condition.

43. A method according to claim 42, wherein the malignant neoplastic condition is a systemic neoplastic malignancy.

44. A method according to claim 43, wherein the systemic neoplastic malignancy is selected from the group consisting of leukaemia such as acute leukemia (AL) , chronic leukemia (CL) , T-cell acute leukemia (T-ALL) , B-cell acute leukemia (B-ALL) , T-cell chronic leukemia (T-CLL) , B-cell chronic leukemia (B-CLL) , prolymphocytic leukemia (PLL) , acute undifferentiated leukemia (AUL) , acute myelogenous leukemia (AML) , chronic myelogenous leukemia (CML) , chronic myelomonocytic leukemia (CMML) , acute promyelocytic leukemia (APL) , pre-B-ALL, and pro-B-ALL; lymphoma such as Burkitt's lymphoma (BL) , non-Hodgkins lymphoma (NHL) , Hodgkins lymphoma (HL) , follicular lymphoma (FL) , diffuse large cell lymphome (DLCL) , T-cell lymphoma, B- cell lymphoma; myelodysplasia; and myeloid.

45. A method according to claim 43 or 44, wherein the chromosomal abnormality is selected from the group consisting of: dup(llq23) (dup exon 5-9/2); dup(llq23) (dup exon 5-9/4); inv(16) (pl3;q22) ; t (1;11) (p32;q23) ; t(l;19) (q23;pl3) ; t(10;ll) (pl4;q23) t(10;ll) (pl4;q23) t (10;14) (q24;qll) t (11;17) (q23;q21) t (11; 19) (q23;pl3.1) ; t (11;19) (q23;pl3.3) ; t (12;21) (pl3;q22) t(12;22) (pl3;qll) t(15;17) (q21;q22) t(15;17) (q21;q22) 16;21) (pll;q22) ; 17; 19) (q22;pl3) ;

3) (p21;q26) ;

5) (p23;q35) ;

21) (q26;q22) ;

3) (q21;q26) ;

5) (q25.1;q34)

11) (q21;q23) ;

12) (q33;pl3) ;

17) (q35;q22)

11) (q27;q23) ;

9) (p23;q34) ;

10) (q35;q24) ;

9) (q34;q32) ;

21) (q22;q22)

11) (q22;q23)

12) (q34;pl3)

22) (q34;qll)

11) (ql3;q23) ; and tall^d1"3 (40 kb deletion) , or wherein the chromosomal abnormality is selected from the genes in the group CBFβ/MYHll, SIL1/TAL1, MLLl, EVI-1, MLLl/AFXl, MLLl/AFlp, MLLl/AFlq, E2A/PBX1, E2A/HLF, EVI1, NPM/ALK, NPM/MLF, AMLl/EVIl, MLLl/- AF4, TEL/PDGf/5, NPM/RARce, DEK/CAN, SET/CAN, MLL1/AF6, HOXll, AML1/MTG8, MLL1/AF9 , BCR/ABL, MLL1/AF10, MLL1/AF17, PLZF/- RARc, MLL/ELL, MLL/ENL, TEL/AML 1, PML/RARα, FUS/ERG, AMLl/ - MDS, AML1/EAP, TEL/MN1, MLL exon 5-9/2, and MLL exon 5-9/4.

46. A method according to claim 42, wherein the malignant neoplastic condition is a non- systemic neoplastic malignancy.

47. A method according to claim 46, wherein the non- systemic neoplastic malignancy is selected from the group consisting of carcinoma, adenocarcinoma, liposarcoma, fibrosarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, glioma, neuroblastoma, medullablastoma, malignant melanoma, neurofibrosarcoma, heamangiosarcoma, lymphangiosarcoma, malignant teratoma, dysgerminoma, seminoma, and choriocarci- noma.

48. A method according to claim 47, wherein the carcinoma is " selected from carcinoma of the breast, bronchus, colorectum, stomach, prostate, ovary, lymphoid tissue, lymphoid marrow, uterus, pancreas, esophagus, urinary bladder, kidney, or skin.

49. A method according to claim 47, wherein the malignant neoplastic condition is selected from the group consisting of papillary thyroid carcinoma, Ewing's sarcoma, liposarcoma, rhabdomyosarcoma, synovial sarcoma, and melanoma of soft parts.

50. A method according to any of the preceding claims, wherein the sample of nucleic acids is derived from cells of the bone marrow in the subject.

51. A method according to any of claims 1-49, wherein the sample of nucleic acids is derived from peripheral blood cells in the subject.

52. A method according to any of claims 1-49, wherein the sample of nucleic acids is derived from placental cells.

53. A method according to any of claims 1-49 wherein the sample of nucleic acids is derived from foetal cells.

54. A method according to claim 1-49, wherein the sample of nucleic acids is derived from amniotic fluid.

55. A method according to any of the preceding claims, wherein at least one of the primers used in the multiplex molecular amplification procedure is labelled.

56. A method according to claim 55, wherein the label is selected from the group consisting of a radioactive label, a coloured label, a fluorescent label, a biotinyl label, a phosphate label, an amin label, and a tiol label.

57. A method according to any of the preceding claims, wherein the sample of nucleic acids is subjected to at least two multiplex molecular amplification procedures as defined in any of the preceding claims .

58. A method according to claim 57, wherein the at least two multiplex molecular amplification procedures are carried out in parallel.

59. A method according to claim 57 or 58, wherein the at least two multiplex molecular amplification procedures are carried out under substantially the same conditions with respect to physical parameters and timing.

60. A method according to any of the preceding claims, where- in the presence or absence of the characteristic sequences is determined by means of gel electrophoresis, sequence analysis, HPLC, FPLC and by flouresence spectofotometri .

61. A method according to any of claims 2-60, wherein the nucleic acid fragment of the internal standard is a cDNA molecule derived from the subject.

62. A method according to claim 61, wherein the cDNA molecule is obtained by use of specific or non-specific cDNA primers in a molecular amplification procedure wherein the templates in the procedure are in the form of mRNA derived from the subject.

63. A method according to claim 62, wherein the cDNA molecule is obtained in the molecular amplification procedure defined in claim 27.

64. A method according to claim 62, wherein the cDNA molecule corresponds to a constitutively expressed DNA fragment.

65. A method according to any of the preceding claims, wherein primers used in the molecular amplification procedure are so constructed, that

1) they hybridize to their respective target sequences at or below substantially the same temperature,

2) they are substantially specific for their respective target sequences,

3) they exhibit substantially no intramolecular hybridization,

4) they have a higher melting point in the 5' -end than in the 3 ' -end,

5) no two primers are, in the molecular amplification procedure, capable of together initiating and sustaining amplification of nucleic acid fragments in the sample which corre- spond to normally occurring sequences not associated with a condition in the subject,

6) no primer contains more than 5 consecutive guanidyl residues,

7) they exhibit substantially no intermolecular hybridiza- tion.

66. A method according to claim 65 wherein the primers hybridize to their respective target sequences at a temperature difference within 5°C.

67. A method according to any of claims 65 and 66 wherein the primers are complementary to their respective sequence.

68. A method according to any of claims 65-67 wherein the primers have a delta G being above -1 within the primer.

69. A method according to any of claims 65-68 wherein the primers have a higher melting point in the 5' -end than in the 3' -end of at least 1°C, preferable at least 6°C.

^'70. A method according to any of claims 65-69 wherein no primer contains more than 3 consecutive guanidyl residues.

71. A method according to any of claims 65-70 wherein the primer dimer has a delta G being above -10.

72. A method according to any of the preceding claims, wherein the primers used in the molecular amplification procedure have nucleic acid sequences which are selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 182.

73. A method according to any of the claims 27-72, wherein the cDNA primers are selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 32 and SEQ ID NO: 178 through SEQ ID NO: 182.

74. A method according to any of the preceding claims wherein the sample material is 1 μg nucleic acid for each multiplex molecular amplication procedure.

75. A nucleic acid fragment which has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 through

SEQ ID NO: 182.

76. A kit comprising 7 mutually distinct primers selected from the group of cDNA primers consisting of SEQ ID NO: 1 through SEQ ID NO: 32 and SEQ ID NO: 178 through SEQ ID NO: 182; and of PCR primers selected from SEQ ID NO: 33 trough SEQ ID NO: 177.

77. A kit according to claim 77 wherein the primers are attached to a device selected from the group comprising a well, a capillary tube, a stick, and a bead.