CA1339731C - Multiplex genomic dna amplification for deletion detection - Google Patents

Abstract

The present invention relates to a method for detecting multiple DNA sequences simultaneously. The method involves amplification of multiple sequences simultaneously by annealing a plurality of paired oligonucleotide primers to single stranded DNA. One member of each pair is complementary to the sense strand of a sequences and the other member is complementary to a different segment of the anti-sense strand of the same sequence. The amplification occurs by alternately annealing and extending the primers. The invention also includes oligonucleotide primer sequences helpful in detecting genetic diseases and/or exogenous DNA sequences.

Description

Multiplex Genomic DNA Amplification for Deletion Detection ts Field of the Invention This invention relates to the field of simultaneous detection of deletions in genomic DNA
sequences by the process of amplification of multiple sequences within the hemizygous or homozygous genome. The nucleic acid sequences are amplified by the process of simultaneous multiple repetitive reactions. This method of deletion detection is useful in a variety of areas including screening for genetic disease, and animal husbandry. Multiplex DNA amplification is also applicable to the simultaneous analysis of multiple genomic sequences and is useful in forensic medicine, disease screening, and in the development of recombinant or transgenic organisms.

Backqround This invention is an improvement on currently established procedures for the detection of genetic 133~731 diseases resulting from mutations and deletions in genomic DNA sequences. Prenatal diagnosis and carrier detection of many X-linked diseasesare available via Southern analysis using full length cDNA clones. Unfortunately, there are several major limitations that prevent widespread and routine use of Southern analysis for diagnosis of genetic disease. In many of the X-linked diseases, the defective sequences are unknown and probes are unavailable. In other diseases, such as X-linked ~ muscular dystrophy, there are multiple exons, at least 60, scattered over a large area of genomic DNA, appro~imately 2.4 million bases. The introns average 35 Kb in length.
In the case of muscular dystrophy, at least 7-9 separate 15 cDNA subclones are necessary for Southern blot analysis to resolve each e~on-containing restriction fragment for hyplotype assignment or diagnosis of genomic alterations.
Furthermore, Southern analysis is an e~pensive, tedious, and time-consuming technique that requires the use of radioisotopes, making it unsuitable for routine use in clinical laboratories.
An alternative to Southern analysis for mutation and deletion detection is the polymerase chain reaction (PCR) described by Mullis et al. in U. S. Patent No.
4,683,195 which issued on July 28, 1987 and by Mullis in U. S. Patent No. 4,683,202 which issued on July 28, 1987.
With PCR, specific regions of a gene can be amplified up to a million-fold from nanogram quantities of genomic DNA. After amplification the nucleic acid sequences can be analyzed for the presence of mutant alleles either by direct DNA sequencing or by hybridization with allele-specific oligonucleotide probes. The PCR technique has proven useful in the diagnosis of several diseases including ~-thalassemia, hemophilia A, sickle cell anemia and phenylketonuria. Routine screening for genetic diseases and exogenous DNA sequences, such as virus, with , .
'.''~', -3- 1~9~31 PCR, has been limited by the ability to conduct tests for only a single sequence at a time. Screening for a plurality of possible DNA sequences requires a - 5 cumbersomely large number of separate assays, thus increasing the time, expense, and tedium of performing such assays. For e~ample, in some diseases, such as Duchenne muscular dystrophy (DMD), PCR diagnosis has been limited since point mutations leading to DMD have not been 0 identified. Appro~imately 60% of the cases of DMD are due to deletions. The other 40% are unknown at present, but probably involve mutations of the intron-exon splice sites or the creation of premature stop codons. Thus a large gene like the DMD gene must be screened with multiple assays In both U. S. Patent ~os. 4,683,195 and 4,683,202, procedures are described for amplification of specific sequences. Both patents describe procedures for detecting the presence or absence of at least one specific nucleic acid sequence in a sample containing a mixture of sequences. Although the patents claim at least one sequence and state that multiple sequences can be detected, they do not provide an effective procedure for amplifying multiple sequences at the same time. In the e~amples, single sequences are amplified or multiple sequences are amplified sequentially. Adding primers for a second sequence is usually possible, but when primers for more than two sequences are added the procedure falls apart. The present appli~cation is an improvement on the PCR method and solves the problems encountered when primers for multiple sequences are reacted simultaneously. The present invention describes a procedure for simultaneous amplification of multiple sequences, and for the application of this multiplex =
amplification procedure in order to detect-a plurality of deletions within the same gene or within multiple genes.

~;

_4_ 1339731 The procedures of the present application provide improved methods for the detection of deletions in hemizygous genes on the X and Y chromosomes. The procedures are effec~ive in detecting genetic diseases caused by deletions on the X or Y chromosome, for example, DMD. They are also effective in detecting homozygous deletions and may be used to simultaneously screen for many possible homozygous or hemizygous deletions as long as parts of the appropriate genetic sequences are known.
The procedure for multi~le~ amplification ~lso enables simultaneous analysis of multiple genetic loci regardless of the presence or absence of deletions.

SummarY of the Invention In broad terms, one aspect of the present invention is a method for simultaneously detecting deletions at a plurality of genomic DNA sequences.
Another aspect of the present invention is the detection of X-linked genetic diseases.
A further aspect of the present invention is the diagnosis of DMD.
A further aspect of the present invention is the simultaneous analysis of multiple genetic loci for -polymorphisms and/or non-deletion mutations.
Thus, there is provided in accordance with one aspect of the present invention, a method for detecting deletions from at least three DNA sequences, comprising the steps of:
Treating said DNA to form single-stranded complementary strands;
Adding at least three pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense strand and the other primer 1~3~731 of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand, each primer having similar melt temperatures;
Annealing the primers to their complementary sequences, all primers being subjected to the same reaction conditions;
Simultaneously extending the annealed primers from the 3' terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer;
Separating said extension products from said templates to produce single-stranded molecules, said extension products, after separating from their complement, serving as templates for the synthesis of an extension product from the other primer of each pair;
Amplifying said single-stranded molecules by repeating at least once, said annealing, extending and separating steps;
Identifying said amplified extension products from each different sequence; and Analyzing said amplified extension products to detect known deletions.
Additional embodiments include detection of deletions at a plurality of genomic DNA sequences on the X
and Y chromosomes or on autosomal chromosomes when the deletions are homozygous. A variety of X-linked diseases can be detected including ornithine transcarbamylase deficiency, hypo~anthine phosphoribosyltransferfase deficiency, steroid sulfatase deficiency and X-linked muscular dystrophy.
In another embodiment, X-linked muscular dystrophy is detected using a plurality of paired primers which are complementary to different sequences within the gene coding for the protein dystrophin. Other embodiments include multiple oligonucleotide primers useful in detecting X-linked genetic disease.
Other and further objects, features and advantages will be apparent from the following description , _, ,~

1~.3~731 of the presently preferred embodiments of the invention given for the purpose of disclosure when taken in conjunction with the accompanying drawings.

Brief Discussion of the Drawinqs The invention will be more readily understood from a reading of the following specification and by references to the accompanying drawings, forming a part thereof:
Figure 1 is a schematic representation o~ the DMD
gene illustrating the approximate size of the locus, the position of the amplified fragments and the location of the genomic regions that have been cloned and sequenced.
Figure 2 is an e~ample of a PCR reaction used to detect a deletion in fetal DNA for prenatal diagnosis.
Figure 3 represents the multiple~ DNA
a~plification of lymphoblast DNA from unrelated male DM~
patients. A. and B. show two sets of ten samples. Each 20 DRL # refers to the R.J. Kleberg Center for Human Genetics Diagnostic Research Laboratory family number. MW: Hae III
digested ~Xl74 DNA. (-): no template DNA added to the reaction. The relationship between the amplified region and the region on the gene is indicated to the right of A. The letters correspond to those on Figure l.
Figure 4 represents Multiplex DNA amplification for prenatal diagnosis of DMD. Shown are the results of amplification using DNA from an affected male (AM;
lymphoblast DNA) and a male fetus (MF; cultured amniotic fluid cell DNA) from si~ different families. Both the affected male and the fetal DNAs of DRL #s 521 and 531 display a deletion of region f (Fig. l);diagnosing these fetuses as affected. In DRL ~ 43C the affected male is deleted for all regions except f, while the fetus is unaffected. The affected male in DRL # 483 is deleted for region a, while the male fetus is unaffected. Neither of 7 1~3~731 the samples from DRL #s 485 or 469 displayed a deletion with this technique.
Figure 5 represents Multiplex DNA amplification 5 from chorionic villus specimen (CVS) DNA. Both the affected male (AM; lymphoblast DNA) and the male fetus (MF; CVS DNA) from DRL # 92 display a deletion of regions e and f (Fig. l), diagnosing the fetus as affected. CVS
DNA from DRL # 120 did not display a deletion with this 10 technique.
Figure 6 shows amplification of seven e~on regions of the DMD locus.
The drawings are not necessarily to scale and certain features of the invention may bè exaggerated in scale or shown in schematic form in the interests of clarity and conciseness.

Detailed Description It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein, without departing from the scope and spirit of the invention.
The term Uoligonucleotide primersU as used herein defines a molecule comprised of more than three deoxyribonucleotides or ribonucleotides. Its exact length will depend on many factors relating to the ultimate function and use of the oligonucleotide primer, including temperature, source of the primer and use of the method.
The oligonucleotide primer can occur naturally, as in a purified restriction digest, or be produced synthetically. The oligonucleotide primer is capable of acting as an initiation point for synthesis when placed under conditions which induce synthesis of a primer extension product complementary to a nucleic acid strand.
The conditions can include the presence of nucleotides and an inducing agent such as a DNA polymerase at a suitable 13~9731 temperature and pH. In the preferred embodiment, the - primer is a single-stranded oligodeoxyribonucleotide of sufficient length to prime the synthesis of an extension 5 product from a specific sequence in the presence of an inducing agent. In the deletion detection procedure, the oligonucleotides are usually at least greater than 12 mers in length. In the preferred embodiment, the oligonucleotide primers are about 18 to 29 mers in length. Sensitivity and specificity of the oligonucleotide primers are determined by the primer length and uniqueness of sequence within a given sample of template DNA. Primers which are too short, for example, less than about 12 mer, may show non-specific binding to a wide variety of sequences in the genomic DNA and thus are not very helpful. In the preferred embodiment, the oligonucleotide primer is usually selected for its ability to anneal to intron sequences in the proximity of the 5' or 3' end of the e~on or to anneal to a sequence at the intron-e~on junction. Since the known deletion defects resulting in genetic diseases result from deletions that include the exons or intron-splice site regions, it is preferable to have primers complementary to intron sequences.
Each primer pair herein was selected to be substantially complementary to the different strands of each specific sequence to be amplified. Thus, one primer of each pair is sufficiently complementary to hybridize with a part of the sequence in the sense strand and the other primer of each pair is sufficiently complementary to hybridize with a different part of the same sequence in the anti-sense strand. Thus, although the primer sequence need not reflect the exact sequence of the template, the more closely it does reflect the e~act sequence the better the binding during the annealing stage.

,.

g Within a primer pair, each primer preferably - binds at a site on the sequence of interest distant from the other primer. In the preferred embodiment the 5 distance between the primers should be sufficient to allow the synthesis of an e~tension product between the two binding sites, yet close enough so that the e~tension product of each primer, when separated from its template, can serve as a template for the other primer. The extension products from the two paired primers are complementary to each other and can serve as templates for further synthesis. The further apart the binding sites, the more genomic DNAthere is which--can be sc~eened. However~-if the distance is too great the extension products will not 5 efficiently overlap with the primers and thus amplification will not occur.
As used herein the term ~e~tension product~
refers to the nucleotide sequence which is synthesized from the 3' end of the oligonucleotide primer and which is complementary to the strand to which the oligonuc-leotide primer is bound.
As used herein the term ~di~ferentially labeled"
shall indicate that each extension product can be distinguished from all the others because it has a different label attached or is of a different size or binds to a specifically labelled oligonucleotide. One skilled in the art will recognize that a variety of labels are available. For example, these can include radioisotopes, fluorescers, chemiluminescers, enzymes and antibodies. Various factors affect the choice of the label. These include the effect of the label on the rate of hybridization and binding of the primer to the DNA, the sensitivity of the label, the ease of making the labeled primer, probe or extension products, the ability to 3~ automate, available instrumentation, convenience and the like. For e~ample, a different radioisotope could be used ~ c ~ 9 ~ 3 1 such as 32p, 3H, or C; a different fluorescer such asfluorescein, tetramethylrhodamine, Texas Red or 4-chloro-7- nitrobenzo-2-oxa-l-diazole (NBD); or a mixture 5 of different labels such as radioisotopes, fluorescers and chemiluminescers. Alternatively, the primers can be selected such that the amplified e~tension products for each sequence are of different lengths and thus can be separated by a variety of methods known in the art.
0 Similarily, the e~tension products could include a restriction fragment length polymorphism which could be used to distinguish different e~tension products. In these examples, each primer or its extension product can be differentiated from all the other primers when they are in a mixture. Alternatively, probes which bind to the amplified extension products could be labeled and sets of probes which distinguish alleles of a single sequence within a multiplex DNA amplification reaction may be used whether or not labelled.
Each specific, different DNA sequence, which is to be detected herein, can derive from genomic DNA of the organism or e~ogenous DNA such as virus, bacteria or parasites. The source of genomic DNA from the organism to be tested can be blood, hair or tissue (including chorionic villi, amniotic cells, fibroblasts and biopsies). The source of DNA may be freshly obtained or have been suitably stored for extended periods of time.
The DNA must be of sufficient quality to permit amplification. The genomic DNA can be prepared by a variety of techniques known to one skilled in the art.
As used herein, the term "deletion" refers to those genomic DNA sequences in which one or more nucleic acid base has been deleted from the sequence and thus is no longer present in the gene. The size of the deletion can affect the sensitivity of the amplification 1 3 ~9 731 procedure. Generally, the larger the deletion the larger the sensitlvity.
Any specific known nucleic acid sequence can be detected by the present method. Preferably, at least part of the sequence is deleted from the genome. It is only necessary that a sufficient number of bases at both ends of the sequence be known in sufficient detail to prepare oligonucleotide primers which will hybridize to the different strands of the desired sequence at relative positions along the se~uence.
The oligonucleotide primers may be prepared using any suitable method, for e~ample, phosphotriester: and phosphodiester methods or automated embodiments thereof, the synthesis o~ oligonucleotides on a modified solid support, the isolation from a biological source (restriction endonuclease digestion), and the generation by enzymatically directed copying of a DNA or RNA template.
One embodiment of the present invention is a method for simultaneously detecting deletions from at least three DNA sequences, comprising the steps of: treating said DNA to form single-stranded complementary strands; adding at least three pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand, each primer having similar melt temperatures; annealing the primers to their complementary sequences, all primers being subjected to the same reaction conditions; simultaneously extending the annealed primers from the 3' terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer; separating said extension products from said templates to produce single-stranded molecules, said extension products, after separation from the complement, serving as templates for the synthesis of an extension product from the other primer of each pair;
' .

l~9 731 amplifying said single-stranded molecules by repeating, at least once, said annealing, extending and separating steps;
identifying said amplified extension product from each different sequence; and analyzing said amplified extension product to detect known deletions.
One preferred embodiment of the present invention is a method for detecting deletions at a plurality of genomic DNA sequences, wherein said sequences are selected from a group of sequences on the X and Y chromosomes. It is preferrable to detect hemizygous genes on the X and Y
chromosomes, since this increases the level of sensitivity. When the procedure is used to detect the heterozygous state, it requires quantitative measurement, and thus is much less efficient than detecting the presence or absence of sequences as is done for hemizygous genes. For e~ample, if part of an exon has been deleted the multiple~ amplification method of the present invention will detect this by either failing to produce an oligonucleotide sequence or by production of an oligonucleotide sequence of a different size. Furthermore multiple e~ons can be screened at the same time. Thus, it is easy to detect the presence of a deletion. However, in looking at heterozygous states, where the chromosomes have one normal gene and one deleted gene, the normal gene will produce a normal product, and thus there is the necessity to measure the quantitative difference in the production of extension products.
A second embodiment of the present invention is to permit simultaneous amplification of multiple, possibly unrelated sequences for the purpose of their sim-ult~neous analysis. Such analysis may simply involve the determination of whether exogenous sequences (virus, bacteria or other parasites) are present within a sample of DNA, or might involve the detection of polymorphisms or mutations within a plurality of sequences. The .~ . . ., ~ .

1~3~731 polymorphisms or mutations can be detected by a variety of methods well known to those skilled in the art. The methods include, but are not limited to, direct DNA
5 sequencing, allele-specific oligonucleotide hybridization, and competitive oligonucleotide priming.
The multiplex genomic DNA amplifica~on method is preferably used to detect X-linked diseases resulting from deletions in the genomic DNA sequence. Genetic diseases 0 can be caused by a variety of mechanisms including mutations and deletions. The procedure described herein was developed for detection of genetic diseases which result from deletions within the genome. Examples of some X-linked diseases which are candidates for the use of multiple~ genomic DNA amplification are ornithine transcarbamylase deficiency, hypo~anthine phosphoribosyltransferase deficiency, steroid sulfatase deficiency and X-linked muscular dystrophy. Other disorders on the X chromosome or genes on the Y chromosome 20 can also be easily detected. The procedure is also applicable to the detection of any set of known point mutations within a set of genomic sequences. The procedure is also applicable to the simultaneous detection of any set of e~ogenous DNA sequences in a given DNA
sample. The procedure is also applicable to the simultaneous detection of any set of polymorphic or variable tandemly repetitive sequences within a genone.
The advantages of the multiplex amplification system are that numerous diseases or specific DNA sequence alterations can be detected in the same assay. For example, primers to hypoxanthine phosphoribosyltransferfase deficiency, steroid sulfatase deficiency, X-linked muscular dystrophy, ornithine transcarbamylase deficiency and other X-linked diseases can all be run simultaneously on the same sample.
Furthermore, the multiplex amplification procedure is 1~;3'~31 useful for very large genes with multiple exons, such as the dystrophin gene. Because of the large size of the dystrophin locus, Mullis type PCR amplification is not 5 able to scan the whole gene in one assay. Thus, it is necessary for multiple site amplification within the gene to detect all possible deletions which could result in disease. Deletions at the DMD locus can encompass any of the appro~imately 60 plus esons which are distributed over more than 2 million bases of DNA. Virtually all of these exons are separated by large i~trons and so up to 60 separate reactions could be required for complete analysis of DMD deletions. To simplify this task, the present invention of a multiplex genomic DNA amplification for deletion detection can be employed to perform simultaneous examination ~f multiple esons. For example, oligonusleatide primers flanking separate DMD gene exons can be synthesized and combined and used for multiplex DNA
applications. At present, up to at least 7 different DMD
gene sequences have been examined simultaneously. The entire procedure for the multiplex amplification from start-up to photography of the results takes less than 5 hours. The relative locations of the amplified regions do not affect the results and exons have been amplified which have been separated by at least l000 kb. The PCR
amplification technique of Mullis is adequate for one and possibly two pair of primers, but when greater than two pairs of primers are used the procedure will not adequately amplify all the appropriate sequences.
One skilled in the art readily appreciates that as more exon gene sequences become available the applicability of this test will expand to examine for deletions in multiple genes at the same time or examine multiple sites within the same gene at the same time. The later example is important for genes such as dystrophin which are so large that primers annealed to the ends of -15- ~ 31 the gene will not traverse the whole gene sequence. Thus the necessity of doing multiple analysis to detect deletions in different regions of the gene. In addition, 5 as specific mutations within multiple unrelated genes become known, multiplex DNA amplification can be applied to simultaneously assay for the presence of any of these mutations.
Furthermore, as specific or highly variable DNA
0 seguence polymorphisms become known in various genetic Loci, ~u}tiplex DNA amplification can be used to simultaneously analyze these polymorphisms to determine the haplotype or to determine the identity or source of D~A (genetic footprinting).
The number of analyses which can be run simultaneously is unlimited, however, the upper limit is probably about 2~ and is dependent on the size differences required for resolution ~nd~or the ~umber of labels or methods which are available to resolve the extension products. The ability to simultaneously amplify only 9 exons would allow the detection of greater than 90% of all known DMD deletions in a single reaction. The ability to simultaneously amplify even as few as 10 exons allows the rapid and simple diagnosis of DMD deletions using only a few separate reactions. Assuming that there are about 60 exons in the DMD gene and that the exons are widely separated such that primers are needed for every exon, a maximum of 6 separate assays is needed to detect all deletions in this gene. Under the same assumptions the Mullis PCR method would require 60 separate reactions to detect the deletions in this gene. Thus, as the size of the gene increases and the number of exons which cannot be detected together increases the advantages of this method are greatly enhanced. Furthermore, use of an automatic PCR apparatus (such as that produced by Perkin-Elmer/Cetus) and DNA sequencing machines will facilitate resolution and detection of amplified DNA
fragments, will help automate the assay and will lead to the method being applied routinely in clinical 5 laboratories without the need for highly trained research personnel.
The following examples are offered by way of illustration and are not intended to limit the invention in any manner. In the examples all percentages are by 0 weight, if for solids and by volume if for liquids, and all temperatures are in degrees Celsius u~less otherwise noted.

The following conditions are currently in use to perform simultaneous amplification of a plurality of separate ge~omiC regions within the human DMD gene. These conditions may need to ~e slightly modified depending on the particular regions to be amplified, the number and length of sequences to be amplified, and the choice of oligonucleotide primers. The time of reaction is highly dependent on the overall sequence length. Thus, as the number of amplified sequences increase and/or the length of amplified sequences increases, the time must be increased. The temperature is dependent on the length, the uniqueness of the primer sequence and the relative percentage of GC bases. The longer the primers, the higher the temperature needed. The more unique the sequence, the lower the temperature needed to amplify. GC
rich primers need higher temperatures to prevent cross hybridization and to allow unique amplification. However, as the AT percentage increases, higher temperatures cause these primers to melt. Thus, these primers must be lengthened for the reaction to work.
Template DNA was prepared from the tissue chosen for analysis using a variety of well-established methods -17- ~ 3~31 known to those skilled in the art. Typically, 100 111 reaction volumes were uti lized . Appro~imately 500 ng of DNA was added to a solution comprised of the foi lowing:
a pair of primers specific for a targeted genomic region within human s DMD gene, each in an amount of 1~4M final concentration; a mixture - of all four dNTPs in the range of O.SmM - 20mM final concentration, 67 n~I Tris-HCL tpH 8.8 at 25~]; 6.7 mM magnesium chloride;
16. 6 snM ammonium sulfate; 10 mM B-mercaptoethanol;

6 . 7 ~M ethylene diamine tetra-acetic acid (EDTA); and 10 170 ~g/mL bovine serum albumin. This solution can be prepared beforehand and appears to be stable for very long periods of storage at -70~. The enzyme, Taq polymerase, was added to achieve a f inal concentration of 100 units/mL. This reaction misture was gently mi~ed. The 15 reaction mi~ture was overlaid with about 50 ~L of paraffin oil, and the reaction vessel (preferably a 0.5 ml microcentrifuge tube) was centrifuged at 14, 000 :~: g for 10 sec. Amplificatic~n was performed either by manually tran5ferring the reaction vessels between glycerol filled 20 heat blocks at the appropriate temperatures, or automatically transferring the reaction vessels with a Perkin-Elmer/Cetus G~orporation thermocycler using- the ' step-cycle ' functions . The reaction was controlled by regulated and repetitive temperature changes of various 25 duration. Initially the reaction was heated to 94~ for 7 minutes. Subsequently 25 cycles of the following temperature durations were applied: 94~ for 1 minute, then -~
55~ for 45 seconds, then 65~ for 3 1/2 minutes. Following completion of the f inal cycle the~- reaction was incubated 30 at 65~ for an additional 7 minutes. Reactions were then stored at 4~ until analysis.
Genomic DNA deletions and/or exogenous DNA
sequences were determined by examining the amplification products. For e2cample, the- lack of an expected 35 amplification product indicates a deletion. Many methods for this determination are known to those skilled in the art. The preferred method involves electrophoresis of about one-twentieth of the reaction on a 1.4~ (weight/vol) 3 l agarose gel in the following buffer: 40 mM tris-HCl;
20 mM sodium acetate, 1 mM EDTA (adjusted to pH 7.2 with glacial acetic acid), and 0.5~g/~1. of ethidium 5 bromide. Electrophoresis was performed at 3.7 volts/cM
for 100 minutes per 14 cM of agarose gel length. Analysis was completed by examining the electrophoresed reaction products on an ultraviolet radiation transilluminator, and the results were photographed for permanent records.
0 When the analysis requires determination of DNA
sequence polymorphisms or mutations within individual amplification products the agarose gel is transferred to an appropriate DNA binding medium such as nitrocellulose usinq well-established procedures, for e~ample, Southern blotting. Individual DNA sequences within the amplified DNA fragments can be determined by a variety of techniques including allele-specific oligonucleotide hybridization.
Alternatively, reaction products may be further analyzed prior to electrophoresis on agarose gel by competitive oligonucleotide primer amplification, using separate allele-specific primers for each amplified DNA sequence of the multiplex amplification reaction products.
A third method for determining DNA sequence differences within individual amplification products does not require electrophoresis. In this method, aliquots of the multiplex amplification reaction are sequentially applied to an appropriate DNA binding membrane such as nitrocellulose, and then each aliquot is analyzed via hybridization with individual members of sets of allele-specific oligonucleotide (ASO) probes, each separate aliquot being hybridized with one member of a pair of ASO probes specific for one member of the multiply amplified DNA sequences.

Figure l is a schematic representation of the DMD
locus. The relative location of the exons used in the DMD
5 gene amplification examples are illustrated.
For detection of DMD, a variety of probes can be used either in individual PCR reactions or in combinations in multiplex PCR reactions. These probes are shown in Table 1.

1~39731 ~ ~, ~ o~ a' ~ o c~ ~., ~., ~., oo a u, a) ~ o~~ o\~ o\~ o\~ o\O o\O o\O
a~ ~ . . . . . . 0~o ~ ~ o ~r 0 0 ~ 0 '~
.,, a ~ ~ ~ ~ ~
.
~ ~ ~ ~ ~ ~ ~ ~ C~ ~
o a) Q Q Q Q Q Q Q ~ n O Q
.L) ~ O ~D c~ 0 Q. c~
-1 ¢ r~ ~n ~o ~ U cr_ ~ ~
~, r~r~ r) U U ~ r~U c'~
F~ ¢ r~ U CJ ¢ E~ ¢ ~ U ¢ r ~5~ U
r~ ¢ ~ _~~) r~ ~) U ~ cS r~~ rJ cn ~
~ cr~ ~ ~rJ f~,r,~ c~ r~ S ~ ~
X ~ ¢ r~ r~) _ r ~ r I. J ~ r~ r~) J rJ
¢ ~ S r) _ r~ r~ rJ ~ E- ~
Q. ~~3 -E~ rj ~ ~ ¢ J ~ r _:r ,c~
rrJ J E- ~ ~ r -~ rJ ~ , r~ C
r~ ~ J c~ n , I a ~ r~ ~ ~ r~E~
r ~ ~> J - ~U rr rJ - ~ 1.¢ ~ 3 ¢ _~ ~ r~ ~ W i ~
U~ J ~ r~ rJ ~ _ ~ ~ r3 r~
r ~ 3~ ~ ~ .S
a)r ~ ¢ r~ ~ u ~ c'~ 5r~ r~
3 ~ ~ r~ r~ r~ ¢ r~r,~ r~
a ~,~ - ¢r~) r_r ~SrJ ,c~ r~ ~r,~ ~ ~
r) ~ E~ r cr_ _) c~ r a P~ J~S r~ ¢ c~ rr.
J J ~ _)_ r,~ ~ ,¢ r ~ ~ r ~) r3 ~) ~ 1 U ~ ~ r c~
O ~ r~ ¢ ~ ~ ¢ ~ U ~ ~ c'~ U
,~ r~r~ "~ c~ U cr~ r ) r r cd I I I I I I I I I I I II I ~ c~
c~ o k~ H ' r~ H
H ~ ,~ ~ C~
~ H H ~ g ,~ H H -r~
a) ~c ) ,~ ,~
Q ~ ,~
Q N ~ 1 X bl) ~ ~r~ ~t~ ~ r~ ~ 0 ~~ ~ ,~
re~ UJao ~r~l Q~ r-l ~ p~ r-l ~
Q Q a, Q Q Q Q ~ Q Q _ ~, ~~ r.~ 0 ~ Q ~ CO ~ ~ ~ O~
~:o 0o 1' o 0 ,~ t~ ~ Ino L~
r~X r~ ~C 0 ~ r~ ~ r~ ~ r~~4 r~
O '~ r~ ~~1 ~' O
o E- ~g X ~ ~ . . . . . *
Q r~) ~rJ a~
-t f - --21- ~ ~q~ 3 1 In Table 1 each exon is designated a, b, c, d, e, f, or g and corresponds to the same letter in Fig. 1.
When the exon number is known it is listed. If the exon number is not known, the size of the genomic Hind III
fragment containing that exon is listed. Also shown is the size of the exon in base pairs (bp). The PCR primer sequences are shown in 5'-3' orientation. The forward primer (F), hybridizes 5' of the exon, and the reverse primer (R), hybridizes 3' of the exon. The size of the amplified fragment obtained with each primer set is also shown.
The percentage of analyzed DMD patients that are deleted for each indicated exon is shown in column four.
This total number is less than the sum of the individual exon deletion frequencies because many deletions encompass multiple exons.
In Table 2 are the e~on and flanking intron sequences for Exon 17. The exon is from 227 to 402. The primer sequences used to amplify this sequence are 7 to 33 and 396 to 421.

5' 10 20 30 40 50 TAAATTGACT TTCGATGTTG AGATTACTTT CCCTTGCTAT TTCAGTGAAC

CAAACTTAAG TCAGATAAAA CAATTTTATT TGGCTTCAAT ATGGTGCTAT

TTTGATCTGA AGGTCAATCT ACCAACAAGC AAGAACAGTT TCTCATTATT

TTCCTTTGCC ACTCCAAGCA GTCTTTACTG AAGTCTTTCG AGCAATGTCT

GACCTCTGTT TCAATACTTC TCACAGATTT CACAGGCTGT CACCACCACT

CAGCCATCAC TAACACAGAC AACTGTAATG GAAACAGTAA CTACGGTGAC

CACAAGGGAA CAGATCCTGG TAAAGCATGC TCAAGAGGAA CTTCCACCAC

CACCTCCCCA AAAGAAGAGG CAGATTACTG TGGATTCTGA AATTAGGAAA

AGGTGAGAGC ATCTCAAGCT TTTATCTGCA AATGAAGTGG AGAAAACTCA

TTTACAGCAG TTTTGTTGGT GGTGTTTTCA CTTCAGCAAT ATTTCCAGAA

~33~7~1 TCCTCGGGTA CCTGTAATGT CAGTTAATGT AGTGAGAAAA ATTATGAAGT

ACATTTTAAA ACTTTCACAA GAAATCACTA TCGCAACAGA AACTAAATGC

AGGTGATAAA CTACTGTGGT AGCATTTTAA TCCTAAAAGT TTCTTTCTTT

C'1"L'1''1'-L'L'1''1''L TTTCTTCCTT ATAAAGGGCC TGCTTGTTGA GTCCCTAGTT

TTGCATTAAA TGT~ Ll~ TTCCAGTAAC GGAAAGTGCA TTTTCATGAA

860 870 880 890 goa GATGTCTCAG TCTGCGCATC CTAAATCAGG AGTAATTACA GAACACATTT

CCTGTTCTTT GATATTTATA AAGTCTTATC TTGAAGGTGT TAGAATTTTT

AACTGATCTT TTTGTGACTA TTCAGAATTA TGCATTTTAG ATAAGATTAG

GTATTATGTA AATCAGTGGA TATATTAAAT GATGGCAATA A-3' In Table 3 is the eson and flanking intron sequences for E~on d of TahLe 1 [or, the exon located on a 4.1 ~b Hind III fragment]. The e~on is from 295 to 442.
20 The primer sequences used to amplify this sequence are 269 to 293 and 512 to 536.

5' 10 20 30 40 50 TGTCCAAAAT AGTTGACTTT CTTTCTTTAA TCAATAAATA TATTACTTTA

AAGGGAAAAA TTGCAACCTT CCATTTAAAA TCAGCTTTAT ATTGAGTATT

TTTTTAAAAT GTTGTGTGTA CATGCTAGGT GTGTATATTA ATTTTTATTT

GTTACTTGAA ACTAAACTCT GCAAATGCAG GAAACTATCA GAGTGATATC

TTTGTCAGTA TAACCAAAAA ATATACGCTA TATCTCTATA ATCTGTTTTA

CATAATCCAT CTATTTTTCT TGATCCATAT GCTTTTACCT GCAGGCGATT

TGACAGATCT GTTGAGAAAT GGCGGCGTTT TCATTATGAT ATAAAGATAT

TTAATCAGTG GCTAACAGAA GCTGAACAGT TTCTCAGAAA GACACAAATT

CCTGAGAATT GGGAACATGC TAAATACAAA TGGTATCTTA AGGTAAGTCT

TTGATTTGTT TTTTCGAAAT TGTATTTATC TTCAGCACAT CTGGACTCTT

~' -23- ~3~973~

TAACTTCTTA AAGATCAGGT TCTGAAGGGT GATGGAAATT ACTTTTGACT

GTTGTTGTCA TCATTATATT ACTAGAAAGA AAA-3' 5In Table 4 is the exon and flanking intron sequences for Exon e of Table-l Lo . 5 ~b Hind III fragment exon]. The exon is from 396 to 571. The primer sequences used to amplify this sequence are 51 to 75 and 572 to 597.

5' 10 20 30 40 50 AAACATGGAA CATCCTTGTG GGGACAAGAA ATCGAATTTG CTCTTGAAAA

GGTTTCCAAC TAATTGATTT GTAGGACATT ATAACATCCT CTAGCTGACA
160 170 180 l9Q 200 AGCTTACAAA AATAAAAACT GGAGCTAACC GAGAGGGTGC LLl-LLLcccT

GACACATAAA AGGTGTCTTT CTGTCTTGTA TCCTTTGGAT ATGGGCATGT

CAGTTTCATA GGGAAATTTT CACATGGAGC TTTTGTATTT ~-LLL~Ll-LGc CAGTACAACT GCATGTGGTA GCACACTGTT TAATCTTTTC TCAAATAAAA

AGACATGGGG CTTcA-lllLL GTTTTGCCTT TTTGGTATCT TACAGGAACT

CCAGGATGGC ATTGGGCAGC GGCAAACTGT TGTCAGAACA TTGAATGCAA

CTGGGGAAGA AATAATTCAG CAATCCTCAA AAACAGATGC CAGTATTCTA

CAGGAAAAAT TGGGAAGCCT GAATCTGCGG TGGCAGGAGG TCTGCAAACA

GCTGTCAGAC AGAAAAAAGA GGTAGGGCGA CAGATCTAAT AGGAATGAAA

ACATTTTAGC AGAGLLLllA AGCTT-3' In Table 5 is the e~on and flanking intron sequences for Exon f, Table 1 roverlaps the 1.2 Kb and 3.8 Kb Hind III fragments]. The exon is from 221 to 406.
30 The primer sequences used to amplify this sequence are 26 to 53 and 516 to 541.

S' 10 20 30 40 50 TTTTGTAGAC GGTTAATGAA TAATTTTGAA TACATTGGTT AAATCCCAAC

ATGTAATATA TGTAAATAAT CAATATTATG CTGCTAAAAT AACACAAATC

r , -24- ~ 31 AGTAAGATTC TGTAATATTT CATGATAAAT AACTTTTGAA AATATATTTT

TAAACATTTT GCTTATGCCT TGAGAATTAT TTAC~llLll AAAATGTATT

CTCAAATAAA AGACCTTGGG CAGCTTGAAA AAAAGCTTGA AGACCTTGAA

GAGCAGTTAA ATCATCTGCT GCTGTGGTTA TCTCCTATTA GGAATCAGTT

GGAAATTTAT AACCAACCAA ACCAAGAAGG ACCATTTGAC GTTAAGGTAG
410 420 430 440 450~0 GGGAACTTTT TGCTTTAATA llLllGTCTT TTTTAAGAAA AATGGCAATA

TCACTGAATT TTCTCATTTG GTATCATTAT TAAAGACAAA ATATTACTTG

TTAAAGTGTG GTAAGGAAGA CTTTATTCAG GATAACCACA ATAGGCACAG
560 570 ~8Q 590 600 GGACCACTGC AATGGAGTAT TACAGGAGGT TGGATAGAGA GAGATTGGGC

TCAACTCTAA ATACAGCACA GTGGAAGTAG GAA m ATAG C-3' In Table 6 is the exon and flanking intron sequences for Exon L2. The exon is from 180 to 329. The primer sequences used to amplify this sequence are 27 to 52 and 332 to 357.

5' 10 20 30 40 50 TGAGAAATAA TAGTTCCGGG GTGACTGATA GTGGGCTTTA CTTACATCCT

TCTCAATGTC CAATAGATGC CCCCAAATGC GAACATTCCA TATATTATAA

ATTCTATTGT TTTACATTGT GATGTTCAGT AATAAGTTGC TTTCAAAGAG

GTCATAATAG GCTTCTTTCA AATTTTCAGT TTACATAGAG TTTTAATGGA

TCTCCAGAAT CAGAAACTGA AAGAGTTGAA TGACTGGCTA ACAAAACAGA

AGAAAGAACA AGGAAAATGG AGGAAGAGCC TCTTGGACCT GATCTTGAAG

ACCTAAAACG CCAAGTACAA CAACATAAGG TAGGTGTATC TTATGTTGCG

TGCTTTCTAC TAGAAAGCAA ACTCTGTGTA TAGTACCTAT ACACAGTAAC

ACAGATGACA TGGTTGATGG GAGAGAATTA AAACTTAAAG TCAGCCATAT

TTTAAAAATT ATTTTTACCT AATTG~ ll GCAATCTTTG TTGCCAATGG

CCTTGAATAA GTCCCCTCCA AAATTCAGGT GATTGTATTA GGAGATGGAA

i33~73~

TATTTAAGGG TGAATAATCC ATCAGGGCTC CTCCCTTAAG AATAGGATCA

AGTCCCATAT AAAAGAGGCT TCACACAGTG TTCTCCTATC TCTTGACCCT

CTAACATCTT GATCTTGGAT TTCCCAAACT CGAGAACTGT GAAAAAATAA

AGGTACATTC TTCCTAAATT ACCTCATTCT CATTTAAACA CACAAAGTGC

ACACATAGCT G-3' In Table 7 is the e~on and flanking intron sequences for the Exon lacated on a I0 Kb Hind III
fragment. The exon is from 1 to 150.

5' 10 20 30 40 50 TTACTGGTGG AAGAGTTGCC CCTGCGCCAG GGAATTCTCA AACAATTAAA

TGAAACTGGA GGACCCGTGC TTGTAAGTGC TCCCATAAGC CCAGAAGAGC

AAGATAAACT TGAAAATAAG CTCAAGCAGA CAAATCTCCA GTGGATAAAG

GTTAGACATT AACCATCTCT ~lCC~l~ACAT GTGTTAAATG TTGCAAGTAT

TTGTATGTAT TTTGTTTCCT GGGTGCTTCA TTGGTCGGGG AGGAGGCTGG

TATGTGGATT GTTGTTTTGT TTTGllLLL-1-3' In Table 8 is the exon and flanking intron sequences for the Exon located on a 1.6 Kb Hind III
25fragment from 512 to 622.

5' 10 20 30 40 50 AAGCTTTGAT ACTGTGCTTT AAGTGTTTAC CCTTTGGAAA GAAAATAATT

TTGACAGTGA TGTAGAAATA ATTATTTGAT ATTTATTTCA AAACAAAATT

TATATCCAAT ACTAAACACA GAATTTTGTA AAACAATAAG TGTATAAAGT

AAAATGAACA TTAGGATTAT TGAGATTATT GTAGCTAAAA CTAGL~LLLA

TTCATATAAA TTATGTTAAT AAATTGTATT GTCATTATTG CATTTTACTT

TTTTGAAAAG TAGTTAATGC CTGTGTTTCT ATATGAGTAT TATATAATTC

~3.~731 AAGAAGATAT TGGATGAATT ~1-1L1LL-LLAA GTTTAATGTG TTTCACATCT

CTGTTTCTTT TCTCTGCACC AAAAGTCACA TTTTTGTGCC CTTATGTACC

ATTTAGTAAT TTTATTGCTA ACTGTGAAGT TAATCTGCAC TATATGGGTT

CTTTTCCCCA GGAAACTGAA ATAGCAGTTC AAGCTAAACA ACCGGATGTG

GAAGAGATTT TGTCTAAAGG GCAGCATTTG TACAAGGAAA AACCAGCCAC

660 670 68~ 690 700 AATGGGTTAT GCTTCGCCTG TTGTACATTT GCCATTGACG TGGACTATTT

ATAATCAGTG AAATAACTTG TAAGGAAATA CTGGCCATAC TGTAATAGCA

GAGGCAAAGC TGT~L11 L1 G ATCAGCATAT CCTATTTATA TA~L1~L~ATC

TTAAGGCTAT TAACGAGTCA TTGCTTTAAA GGACTCATTT CTGTC-3' In Table 9 is the e~on and flanking intron sequences for the E~on located on a 3.1 Kb Hind III
fragment. The exon is from 519 to 751.

20 5~ 103 113 123 133 143 CCCATCTTGT TTTGCCTTTG 'L'L'L'L-L'LCTTG AATAAAAAAA AAATAAGTAA

AATTTATTTC CCTGGCAAGG TCTGAAAACT TTTGTTTTCT TTACCACTTC

CACAATGTAT ATGATTGTTA CTGAGAAGGC TTATTTAACT TAAGTTACTT
253 263 273 283 293~5 GTCCAGGCAT GAGAATGAGC AAAATCGTTT TTTAAAAAAT TGTTAAATGT

ATATTAATGA AAAGGTTGAA TC'L'1''L1CATT TTCTACCATG TATTGCTAAA

CAAAGTATCC ACATTGTTAG AAAAAGATAT ATAATGTCAT GAATAAGAGT

TTGGCTCAAA TTGTTACTCT TCAATTAAAT TTGACTTATT GTTATTGAAA

TTGGCTCTTT AGCTTGTGTT TCTAATTTTT ~LLLLLCTTC 'LL1LLLCCTT

TTTGCAAAAA CCCAAAATAT TTTAGCTCCT ACTCAGACTG TTACTCTGGT

GACACAACCT GTGGTTACTA AGGAAACTGC CATCTCCAAA CTAGAAATGC

CATCTTCCTT GATGTTGGAG GTACCTGCTC TGGCAGATTT CAACCGGGCT

TGGACAGAAC TTACCGACTG GCTTTCTCTG CTTGATCAAG TTATAAAATC

ACAGAGGGTG ATGGTGGGTG ACCTTGAGGA TATCAACGAG ATGATCATCA

AGCAGAAGGT ATGAGAAAAA ATGATAAAAG TTGGCAGAAG TTTTTCTTTA

AGTTCTTAGG CAACTGTTTC TCTCTCAGCA AACACATTAC TCTCACTATT

CAGCCTAAGT ATAATCAGGT ATAAATTAAT GCAAATAACA AAAGTAGCCA

TACATTAAAA AGGAAAATAT ACAAAAAAAA AAAAAAAAAA AAGCCAGAAA

10 CCTACAGAAT AGTGCTCTAG TAATTAC-3' In Table 10 is the exon and flanking intron sequences for the Exon located on a 1.5 Kb Hind III
fragment. The e~on is from 190 to 337.

5' 10 20 30 40 50 ATCTCTATCA TTAGAGATCT GAATATGAAA TACLl~l~AA AGTGAATGAA
60 70 8a 90 100 AALllNNLAA ATTATGTATG GTTAACATCT TTAAATTGCT TALlLL-lAAA

TTGCCATGTT TGTGTCCCAG TTTGCATTAA CAAATAGTTT GAGAACTATG

TTGGAAAAAA AAATAACAAT TTTATTCTTC TTTCTCCAGG CTAGAAGAAC

AAAAGAATAT CTTGTCAGAA TTTCAAAGAG ATTTAAATGA ATTTGTTTTA

TGGTTGGAGG AAGCAGATAA CATTGCTAGT ATCCCACTTG AACCTGGAAA

AGAGCAGCAA CTAAAAGAAA AGCTTGAGCA AGTCAAGGTA ATTTTATTTT

CTCAAATCCC CCAGGGCCTG CTTGCATAAA GAAGTATATG AATCTATTTT

TTAATTCAAT CATTGGTTTT CTGCCCATTA GGTTATTCAT AGTTCCTTGC

TAAAGTGTTT TTCTCACAAC TTTALlLCl L CTTAACCCTG CAGTTCTGAA

CCAGTGCACA TAAGAACATA TGTATATATG TGT~ LG TATTTATATA

TACACACACA CATATTGCAT CTATACATCT ACACATATAG ATGTATAGAT

TCAATATGTC TAAAAATGTA TATAATTCAC AGLllLLATC TTTGATTTGA

ATATTTAAGG GACTGAGACT CACACTCATA TACTTTT-3' -28- ~3~ 731 Prenatal Diagnosis and Detection of DMD Using PCR

An example of prenatal diagnosis with PCR
deletion detection is demonstrated using synthesized oligonucleotide primers (set b, Table 1). This primer set corresponds to the intron sequences flanking Exon 17 of the ~ n DMD gene, a region which has been isolated and sequenced (Table 2).
The results of this analysis are shown in Figure ~2. The PCR products (one-twentieth of the total reaction) were obtained with template DNA isolated from a control male a, the male fetus being diagnosed A, the DMD
carrier mother (O) and an affected male ~rother of the fetus ~. Also shown is a DNA molecular weight standard (MW; Hae III digested ~X174 DNA). The results demonstrate that the affected male carries a deletion of ~on 17, which was not amplified, but that the fetus does not carry the deletion and is therefore unaffected. These results indicate that PCR is useful in the diagnosis of DMD cases containing a deletion involving this exon.

Multiplex Detection An example of multiple~ detection is shown in Figures 3A and 3B.
This analysis was done using si~ primer pairs (sets a-f, Table 1) and the conditions described in Example 1. Automatic rather than manual amplification was performed. These oligonucleotide primers represent the flanking regions of six separate DMD gene exons. They ~3~ 731 were combined into a reaction vial and used for multiplex genomic DNA amplifications. Template DNA was isolated from lymphoblasts (from blood sample). Analysis was by 5 agarose gel electrophoresis.
When non-deleted DNA was used as a template, the six dispersed regions of the DMD gene were simultaneously and specificially amplified (Figure 3A, Sample #534).
Discrete deletions, which were detected with this method, 10 are shown in Figures 3A and 3B. Several DNA samples containing normal, partial or total DMD gene deletions are shown. Figures 3A and 3B also show a DN molecular weight standard (MW: Hae III digested ~X174 DNA), and a negative control (-) where no template DNA was added to t5 the reactions. Figure 3A also indicates which amplified ~NA fragment corresponds to which exon (a-f) of Figure 1.

EX~pT.~ 5 Prenatal Diagnosis Multiplex PCR has been used successfully in several prenatal diagnoses. The conditions are as described above in Example 1. Figure 4 shows Multiplex 25 DNA amplification for prenatal diagnosis of DMD. Shown are the results of amplification using DNA from affected males (AM; lymphoblast DNA) and male fetuses (MF; cultured amniotic fluid cell DNA) from six different families.
Analysis was as described in Example 1. Both the affected 30 male and the fetal DNA of DRL #s 521 and 531 display a deletion of region f (Figure 1). Thus these fetuses were diagnosed as affected. In DRL # 43C the affected male is deleted for all regions except f, while the fetus is unaffected. The affected male in DRL #483 is deleted for region a, while the male fetus is unaffected. Neither of the samples from DRL #s 485 or 469 displayed a deletion 7 ~ 1 with this technique. Thus, if a deletion defect causes DMD in these families it occurred in an untested exon.

Prenatal diaqnosis using ~.~ltiple~ DNA amplification of chorionic villus specimen (CVS~ DNA

Figure 5 demonstrates Multiplex DNA amplification from CVS DNA. Both the affected male (AM; lymphoblast DNA) and the male fetus (MF; CVS DNA) from DRL ~ 92 display a deletion of regions e and f (Fig. l). Thus the fetus was diagnosed as affected. CVS DNA from DRL # 120 did not display a deletion with this technique. Samples were analyzed as described in Example l. These results demonstrate that the multiplex amplification technique works well for prenatal diagnosis when CVS DNA is used as the template for amplification.

Multiplex amplification of seven separate exons of the DMD gene This example demonstrates that seven separate DNA
sequences can be simultaneously amplified using the multiplex amplification technique. Conditions were as described in E~ample l. Primer sets a-g (Table l) were added to the reaction. Thus seven e~on regions of the DMD
gene (Figure l) were amplified (Figure 6).

-31- ~ 9 ~ 3 1 Multiplex DNA amplification for the simultaneous detection of mutations leading to multiple common genetic diseases This example describes how the multiplex amplification technique can be used to simultaneously screen a newborn male for any of the most common mutations leading to DMD, sickle-cell anemia and ~l-antitrypsin deficiency. In this assay any or all of the primers sets listed in Table l can be used for multiplex DNA
amplification to diagnose the majority of possible DMD
gene deletions. Additionally, primer sets can be added to t5 the amplification reaction to identify mutations leading to additional genetic diseases. Other primer sets include:

A. 5'-TGGTCTCCTTAAACC-L~L~11-3' 5'-ACACAACTGTGTTCACTAG-3' These oligonucleotides amplify a 167 bp segment of the human B-globin gene, containing the DNA base that is mutated in Bs (sickle-cell) hemoglobinopathy. The presence or absence of the mutant Bs sequence is then determined either by separate dot blot or Southern blot hybridization of the multiplex amplification reaction with each of two labelled allele-specific oligonucleotide (ASO) probes specific for the normal or Bs sequence. The sequence of these two ASO probes is:

l) Normal: 5'-CTCCTGAGGAGA-3' 2) BS: 5'-CTCCTGTGGAGA-3' If dot blot hybridization is used, a separate application of DNA from the multiplex amplification reaction to a DNA
membrane, such as nitrocellulose, is required for each ~ ~ ~ 9 7 3 :l probe that will be used in the hybridization.
Hybridization of each labelled probe, whether the probes are complementary to individual alleles of a given gene or 5 to separate genes, must be performed individually.
Alternatively and preferably, two aliquots of the amplification reaction are separately electrophoresed on agarose gels and transferred to nitrocellulose or a similar membrane using Southern analysis. Each of the two 10 Southern blots are then hybridized with one member of each labelled set of speci~ic ASO primers. Thus each known mutant or normal allele of each DNA fragment amplified in the multiplex reaction can be determined.
In addition to the above described primer sets the following oligonucleotide primers can also be added to the amplification procedure:

B. 5'-ACGTGGAGTGACGAl~l'~llCCC-3' 5'-GTGGGATTCACCACLlLLCCC-3' These primers produce a 450 bp DNA fragment containing the DNA base change that produces the Z allele of the al-antitrypsin gene and leads to ~l-antitrypsin deficiency. The Z allele and the normal M allele are distinguished from other alleles in the multiplex amplification reaction by hybridization with the ASO
probes:

l) Normal (M)allele:5'-ATCGACGAGAAA-3' 2) Mutant (Z)allele:5'-ATCGACAAGAAA-3' Hybridization analysis is performed in parallel with the B-globin probes as described above.

-33- 1 3 ~ ~ 7 3 1 In addition, the oligonucleotides C. 5'-GAAGTCAAGGACACCGAGGAA-3' 5'-AGCCCTCTGGCCAGTCCTAGTG-3' can also be added to the multiple~ reaction to produce a 340 bp DNA region of the al-antitrypsin qene that contains the DNA base change that produces the S allele and leads to ~l-antitrypsin deficiency. The S allele is distin~uished from other alleles in the multiplex amplification as described above for the Bs and Z
alleles by using the two ASO probes specific for the M and S allele:

Normal (M~allele 5'-ACCTGGAAAATG-3' Mutant (S)allele 5'-ACCTGGTAAATG-3' Using the primers described in Table l and in A, B and C of this e~ample,the common mutations leading to DMD, sickle cell anemia and ~l-antitrypsin deficiency can be simultaneously determined.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well, those inherent therein. The methods,procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be e~emplary, and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention or defined by the scope of the appended claims.
WHAT IS CLAIMED IS:

! ~
,.~-,' .

Claims (17)

1. A method for detecting known deletions from at least three DNA sequences, comprising the steps of:
treating said DNA to form single-stranded complementary strands;
adding at least three pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand, each primer having similar melt temperatures;
annealing the primers to their complementary sequences, all primers being subjected to the same reaction conditions;
simultaneously extending the annealed primers from the 3' terminus of each primer to synthesize an extension product complementary to the strands annealed to each primer;
separating said extension products from said templates to produce single-stranded molecules, said extension products, after separation from their complement, serving as templates for the synthesis of an extension product from the other primer of each pair;
amplifying said single-stranded molecules by repeating, at least once, said annealing, extending and separating steps;
identifying said amplified extension products from each different sequence; and analyzing said amplified extension products to detect known deletions.

2. The method of claim 1 for detecting deletions from at least three genomic DNA sequences, wherein said sequences are selected from the group of known sequences on the X and Y chromosomes.

3 The method of Claim 2 for the detection of X-linked disease, wherein said genomic DNA sequences contain a deletion that causes a genetic disease.

4. The method of Claim 3 for the detection of said X-linked genetic diseases selected from the group consisting of ornithine transcarbamylase deficiency, hypoxanthine phosphoribosyltransferfase deficiency, steroid sulfatase deficlency and X-linked muscular dystrophy.

5. The method of Claim 4 for the detection of X-linked muscular dystrophy, wherein each pair of at least three pairs of primers are complementary to different sequences within the gene coding for the dystrophin protein.

6. The method of Claim 5, wherein at least three pairs of primers are selected from the group consisting of:

(1) 5'-GACTTTCGATGTTGAGATTACTTTCCC-3' (2) 5'-AAGCTTGAGATGCTCTCACCTTTTCC-3', (1) 5'-GTCCTTTACACACTTTACCTGTTGAG-3' (2) 5'-GGCCTCATTCTCATGTTCTAATTAG-3', (1) 5'-AAACATGGAACATCCTTGTGGGGAC-3' (2) 5'-CATTCCTATTAGATCTGTCGCCCTAC-3', (1) 5'-GATAGTGGGCTTTACTTACATCCTTC-3' (2) 5'-GAAAGCACGCAACATAAGATACACCT-3', (1) 5'-CTTGATCCATATGCTTTTACCTGCA-3' (2) 5'-TCCATCACCCTTCAGAACCTGATCT-3', (1) 5'-GAATACATTGGTTAAATCCCAACATG-3' (2) 5'-CCTGAATAAAGTCTTCCTTACCACAC-3', and (1) 5'-TTCTACCACATCCCATTTTCTTCCA-3' (2) 5'-GATGGCAAAAGTGTTGAGAAAAAGTC-3'.

7. The method of Claim 3, wherein said genomic DNA is from fetal tissue.

8. The method of Claim 1 for detecting deletions from at least three genomic DNA sequences, wherein at least three pairs of primers are selected from the group consisting of:
(1) 5'-GACTTTCGATGTTGAGATTACTTTCCC-3' (2) 5'-AAGCTTGAGATGCTCTCACCTTTTCC-3', (1) 5'-GTCCTTTACACACTTTACCTGTTGAG-3' (2) 5'-GGCCTCATTCTCATGTTCTAATTAG-3', (1) 5'-AAACATGGAACATCCTTGTGGGGAC-3' (2) 5'-CATTCCTATTAGATCTGTCGCCCTAC-3', (1) 5'-GATAGTGGGCTTTACTTACATCCTTC-3' (2) 5'-GAAAGCACGCAACATAAGATACACCT-3', (1) 5'-CTTGATCCATATGCTTTTACCTGCA-3' (2) 5'-TCCATCACCCTTCAGAACCTGATCT-3', (1) 5'-GAATACATTGGTTAAATCCCAACATG-3' (2) 5'-CCTGAATAAAGTCTTCCTTACCACAC-3', (1) 5'-TTCTACCACATCCCATTTTCTTCCA-3' (2) 5'-GATGGCAAAAGTGTTGAGAAAAAGTC-3', (1) 5'-TGGTCTCCTTAAACCTGTCTT-3' (2) 5'-ACACAACTGTGTTCACTAG-3', (1) 5'-ACGTGGAGTGACGATGCTCTTCCC-3' (2) 5'-GTGGGATTCACCACTTTTCCC-3', and (1) 5'-GAAGTCAAGGACACCGAGGAA-3' (2) 5'-AGCCCTCTGGCCAGTCCTAGTG-3'.

9. A method according to any one of claims 1 through 8, wherein the DNA sequence is:
5' 10 20 30 40 50 TAAATTGACT TTCGATGTTG AGATTACTTT CCCTTGCTAT TTCAGTGAAC

CAAACTTAAG TCAGATAAAA CAATTTTATT TGGCTTCAAT ATGGTGCTAT

TTTGATCTGA AGGTCAATCT ACCAACAAGC AAGAACAGTT TCTCATTATT

TTCCTTTGCC ACTCCAAGCA GTCTTTACTG AAGTCTTTCG AGCAATGTCT

GACCTCTGTT TCAATACTTC TCACAGATTT CACAGGCTGT CACCACCACT

CAGCCATCAC TAACACAGAC AACTGTAATG GAAACAGTAA CTACGGTGAC

CACAAGGGAA CAGATCCTGG TAAAGCATGC TCAAGAGGAA CTTCCACCAC

CACCTCCCCA AAAGAAGAGG CAGATTACTG TGGATTCTGA AATTAGGAAA

AGGTGAGAGC ATCTCAAGCT TTTATCTGCA AATGAAGTGG AGAAAACTCA

TTTACAGCAG TTTTGTTGGT GGTGTTTTCA CTTCAGCAAT ATTTCCAGAA

TCCTCGGGTA CCTGTAATGT CAGTTAATGT AGTGAGAAAA ATTATGAAGT

ACATTTTAAA ACTTTCACAA GAAATCACTA TCGCAACAGA AACTAAATGC

TTAATGGAAA TGGTGTTTTC TGGGGTGAAA GAAGAAACTA TAGAAACTAT

AGGTGATAAA CTACTGTGGT AGCATTTTAA TCCTAAAAGT TTCTTTCTTT

CTTTTTTTTT TTTCTTCCTT ATAAAGGGCC TGCTTGTTGA GTCCCTAGTT

TTGCATTAAA TGTCTTTTTT TTCCAGTAAC GGAAAGTGCA TTTTCATGAA

GAAGTACACC TATAATAGAT GGGATCCATC CTGGTAGTTT ACGAGAACAT

GATGTCTCAG TCTGCGCATC CTAAATCAGG AGTAATTACA GAACACATTT

CCTGTTCTTT GATATTTATA AAGTCTTATC TTGAAGGTGT TAGAATTTTT

AACTGATCTT TTTGTGACTA TTCAGAATTA TGCATTTTAG ATAAGATTAG

GTATTATGTA AATCAGTGGA TATATTAAAT GATGGCAATA A-3' and fragments thereof, said fragments complementary to the sense and anti-sense strands of the gene coding for dystrophin, said fragments capable of annealing to said strands of the dystrophin gene and amplifying dystrophin sequences.

10. A method according to any one of claims 1 through 8, wherein the DNA sequence is:
5' 10 20 30 40 50 TGTCCAAAAT AGTTGACTTT CTTTCTTTAA TCAATAAATA TATTACTTTA

AAGGGAAAAA TTGCAACCTT CCATTTAAAA TCAGCTTTAT ATTGAGTATT

TTTTTAAAAT GTTGTGTGTA CATGCTAGGT GTGTATATTA ATTTTTATTT

GTTACTTGAA ACTAAACTCT GCAAATGCAG GAAACTATCA GAGTGATATC

TTTGTCAGTA TAACCAAAAA ATATACGCTA TATCTCTATA ATCTGTTTTA

CATAATCCAT CTATTTTTCT TGATCCATAT GCTTTTACCT GCAGGCGATT

TGACAGATCT GTTGAGAAAT GGCGGCGTTT TCATTATGAT ATAAAGATAT

TTAATCAGTG GCTAACAGAA GCTGAACAGT TTCTCAGAAA GACACAAATT

CCTGAGAATT GGGAACATGC TAAATACAAA TGGTATCTTA AGGTAAGTCT

TTGATTTGTT TTTTCGAAAT TGTATTTATC TTCAGCACAT CTGGACTCTT

TAACTTCTTA AAGATCAGGT TCTGAAGGGT GATGGAAATT ACTTTTGACT

GTTGTTGTCA TCATTATATT ACTAGAAAGA AAA-3' and fragments thereof, said fragments complementary to the sense and anti-sense strands of the gene coding for dystrophin, said fragments capable of annealing to said strands of the dystrophin gene and amplifying dystrophin sequences.

11. A method according to any one of claims 1 through 8, wherein the DNA sequence is:
5' 10 20 30 40 50 ACCCAAATAC TTTGTTCATG TTTAAATTTT ACAACATTTC ATAGACTATT

AAACATGGAA CATCCTTGTG GGGACAAGAA ATCGAATTTG CTCTTGAAAA

GGTTTCCAAC TAATTGATTT GTAGGACATT ATAACATCCT CTAGCTGACA

AGCTTACAAA AATAAAAACT GGAGCTAACC GAGAGGGTGC TTTTTTCCCT

GACACATAAA AGGTGTCTTT CTGTCTTGTA TCCTTTGGAT ATGGGCATGT

CAGTTTCATA GGGAAATTTT CACATGGAGC TTTTGTATTT CTTTCTTTGC

CAGTACAACT GCATGTGGTA GCACACTGTT TAATCTTTTC TCAAATAAAA

AGACATGGGG CTTCATTTTT GTTTTGCCTT TTTGGTATCT TACAGGAACT

CCAGGATGGC ATTGGGCAGC GGCAAACTGT TGTCAGAACA TTGAATGCAA

CTGGGGAAGA AATAATTCAG CAATCCTCAA AAACAGATGC CAGTATTCTA

CAGGAAAAAT TGGGAAGCCT GAATCTGCGG TGGCAGGAGG TCTGCAAACA

GCTGTCAGAC AGAAAAAAGA GGTAGGGCGA CAGATCTAAT AGGAATGAAA

ACATTTTAGC AGACTTTTTA AGCTT-3' and fragments thereof, said fragments complementary to the sense and anti-sense strands of the gene coding for dystrophin, said fragments capable of annealing to said strands of the dystrophin gene and amplifying dystrophin sequences.

12. A method according to any one of claims 1 through 8, wherein the DNA sequence is:
5' 10 20 30 40 50 TTTTGTAGAC GGTTAATGAA TAATTTTGAA TACATTGGTT AAATCCCAAC

ATGTAATATA TGTAAATAAT CAATATTATG CTGCTAAAAT AACACAAATC

AGTAAGATTC TGTAATATTT CATGATAAAT AACTTTTGAA AATATATTTT

TAAACATTTT GCTTATGCCT TGAGAATTAT TTACCTTTTT AAAATGTATT

TTCCTTTCAG GTTTCCAGAG CTTTACCTGA GAAACAAGGA GAAATTGAAG

CTCAAATAAA AGACCTTGGG CAGCTTGAAA AAAAGCTTGA AGACCTTGAA

GAGCAGTTAA ATCATCTGCT GCTGTGGTTA TCTCCTATTA GGAATCAGTT

GGAAATTTAT AACCAACCAA ACCAAGAAGG ACCATTTGAC GTTAAGGTAG

GGGAACTTTT TGCTTTAATA TTTTTGTCTT TTTTAAGAAA AATGGCAATA

TCACTGAATT TTCTCATTTG GTATCATTAT TAAAGACAAA ATATTACTTG

TTAAAGTGTG GTAAGGAAGA CTTTATTCAG GATAACCACA ATAGGCACAG

GGACCACTGC AATGGAGTAT TACAGGAGGT TGGATAGAGA GAGATTGGGC

TCAACTCTAA ATACAGCACA GTGGAAGTAG GAATTTATAG C-3' and fragments thereof, said fragments complementary to the sense and anti-sense strands of the gene coding for dystrophin, said fragments capable of annealing to said strands of the dystrophin gene and amplifying dystrophin sequences.

13. A method according to any one of claims 1 through 8, wherein the DNA sequence is:
5' 10 20 30 40 50 TGAGAAATAA TAGTTCCGGG GTGACTGATA GTGGGCTTTA CTTACATCCT

TCTCAATGTC CAATAGATGC CCCCAAATGC GAACATTCCA TATATTATAA

ATTCTATTGT TTTACATTGT GATGTTCAGT AATAAGTTGC TTTCAAAGAG

GTCATAATAG GCTTCTTTCA AATTTTCAGT TTACATAGAG TTTTAATGGA

TCTCCAGAAT CAGAAACTGA AAGAGTTGAA TGACTGGCTA ACAAAACAGA

AGAAAGAACA AGGAAAATGG AGGAAGAGCC TCTTGGACCT GATCTTGAAG

ACCTAAAACG CCAAGTACAA CAACATAAGG TAGGTGTATC TTATGTTGCG

TGCTTTCTAC TAGAAAGCAA ACTCTGTGTA TAGTACCTAT ACACAGTAAC

ACAGATGACA TGGTTGATGG GAGAGAATTA AAACTTAAAG TCAGCCATAT

TTTAAAAATT ATTTTTACCT AATTGTTTTT GCAATCTTTG TTGCCAATGG

CCTTGAATAA GTCCCCTCCA AAATTCAGGT GATTGTATTA GGAGATGGAA

TATTTAAGGG TGAATAATCC ATCAGGGCTC CTCCCTTAAG AATAGGATCA

AGTCCCATAT AAAAGAGGCT TCACACAGTG TTCTCCTATC TCTTGACCCT

CCACCATGCA CCACCATGTG AAAACTCTGT GAAAAGGCCC TCACCAGATG

CTAACATCTT GATCTTGGAT TTCCCAAACT CGAGAACTGT GAAAAAATAA

AGGTACATTC TTCCTAAATT ACCTCATTCT CATTTAAACA CACAAAGTGC

ACACATAGCT G-3' and fragments thereof, said fragments complementary to the sense and anti-sense strands of the gene coding for dystrophin, said fragments capable of annealing to said strands of the dystrophin gene and amplifying dystrophin sequences.

14. A method according to any one of claims 1 through 8, wherein the DNA sequence is:
5' 10 20 30 40 50 TTACTGGTGG AAGAGTTGCC CCTGCGCCAG GGAATTCTCA AACAATTAAA

TGAAACTGGA GGACCCGTGC TTGTAAGTGC TCCCATAAGC CCAGAAGAGC

AAGATAAACT TGAAAATAAG CTCAAGCAGA CAAATCTCCA GTGGATAAAG

GTTAGACATT AACCATCTCT TCCGTCACAT GTGTTAAATG TTGCAAGTAT

TTGTATGTAT TTTGTTTCCT GGGTGCTTCA TTGGTCGGGG AGGAGGCTGG

TATGTGGATT GTTGTTTTGT TTTGTTTTTT-3' and fragments thereof, said fragments complementary to the sense and anti-sense strands of the gene coding for dystrophin, said fragments capable of annealing to said strands of the dystrophin gene and amplifying dystrophin sequences.

15. A method according to any one of claims 1 through 8, wherein the DNA sequence is:
5' 10 20 30 40 50 AAGCTTTGAT ACTGTGCTTT AAGTGTTTAC CCTTTGGAAA GAAAATAATT

TTGACAGTGA TGTAGAAATA ATTATTTGAT ATTTATTTCA AAACAAAATT

TATATCCAAT ACTAAACACA GAATTTTGTA AAACAATAAG TGTATAAAGT

AAAATGAACA TTAGGATTAT TGAGATTATT GTAGCTAAAA CTAGTGTTTA

TTCATATAAA TTATGTTAAT AAATTGTATT GTCATTATTG CATTTTACTT

TTTTGAAAAG TAGTTAATGC CTGTGTTTCT ATATGAGTAT TATATAATTC

AAGAAGATAT TGGATGAATT TTTTTTTTAA GTTTAATGTG TTTCACATCT

CTGTTTCTTT TCTCTGCACC AAAAGTCACA TTTTTGTGCC CTTATGTACC

AGGCAGAAAT TGATCTGCAA TACATGTGGA GTCTCCAAGG GTATATTTAA

ATTTAGTAAT TTTATTGCTA ACTGTGAAGT TAATCTGCAC TATATGGGTT

CTTTTCCCCA GGAAACTGAA ATAGCAGTTC AAGCTAAACA ACCGGATGTG

GAAGAGATTT TGTCTAAAGG GCAGCATTTG TACAAGGAAA AACCAGCCAC

TCAGCCAGTG AAGGTAATGA AGCAACCTCT AGCAATATCC ATTACCTCAT

AATGGGTTAT GCTTCGCCTG TTGTACATTT GCCATTGACG TGGACTATTT

ATAATCAGTG AAATAACTTG TAAGGAAATA CTGGCCATAC TGTAATAGCA

GAGGCAAAGC TGTCTTTTTG ATCAGCATAT CCTATTTATA TATTGTGATC

TTAAGGCTAT TAACGAGTCA TTGCTTTAAA GGACTCATTT CTGTC-3' and fragments thereof, said fragments complementary to the sense and anti-sense strands of the gene coding for dystrophin, said fragments capable of annealing to said strands of the dystrophin gene and amplifying dystrophin sequences.

16. A method according to any one of claims 1 through 8, wherein the DNA sequence is:
5' 103 113 123 133 143 CCCATCTTGT TTTGCCTTTG TTTTTTCTTG AATAAAAAAA AAATAAGTAA

AATTTATTTC CCTGGCAAGG TCTGAAAACT TTTGTTTTCT TTACCACTTC

CACAATGTAT ATGATTGTTA CTGAGAAGGC TTATTTAACT TAAGTTACTT

GTCCAGGCAT GAGAATGAGC AAAATCGTTT TTTAAAAAAT TGTTAAATGT

ATATTAATGA AAAGGTTGAA TCTTTTCATT TTCTACCATG TATTGCTAAA

CAAAGTATCC ACATTGTTAG AAAAAGATAT ATAATGTCAT GAATAAGAGT

TTGGCTCAAA TTGTTACTCT TCAATTAAAT TTGACTTATT GTTATTGAAA

TTGGCTCTTT AGCTTGTGTT TCTAATTTTT CTTTTTCTTC TTTTTTCCTT

TTTGCAAAAA CCCAAAATAT TTTAGCTCCT ACTCAGACTG TTACTCTGGT

GACACAACCT GTGGTTACTA AGGAAACTGC CATCTCCAAA CTAGAAATGC

CATCTTCCTT GATGTTGGAG GTACCTGCTC TGGCAGATTT CAACCGGGCT

TGGACAGAAC TTACCGACTG GCTTTCTCTG CTTGATCAAG TTATAAAATC

ACAGAGGGTG ATGGTGGGTG ACCTTGAGGA TATCAACGAG ATGATCATCA

AGCAGAAGGT ATGAGAAAAA ATGATAAAAG TTGGCAGAAG TTTTTCTTTA

AAATGAAGAT TTTCCACCAA TCACTTTACT CTCCTAGACC ATTTCCCACC

AGTTCTTAGG CAACTGTTTC TCTCTCAGCA AACACATTAC TCTCACTATT

CAGCCTAAGT ATAATCAGGT ATAAATTAAT GCAAATAACA AAAGTAGCCA

TACATTAAAA AGGAAAATAT ACAAAAAAAA AAAAAAAAAA AAGCCAGAAA

CCTACAGAAT AGTGCTCTAG TAATTAC 3' and fragments thereof, said fragments complementary to the sense and anti-sense strands of the gene coding for dystrophin, said fragments capable of annealing to said strands of the dystrophin gene and amplifying dystrophin sequences.

17. A method according to any one of claims 1 through 8, wherein the DNA sequence is:
5' 10 20 30 40 50 ATCTCTATCA TTAGAGATCT GAATATGAAA TACTTGTCAA AGTGAATGAA

AATTTNNTAA ATTATGTATG GTTAACATCT TTAAATTGCT TATTTTTAAA

TTGCCATGTT TGTGTCCCAG TTTGCATTAA CAAATAGTTT GAGAACTATG

TTGGAAAAAA AAATAACAAT TTTATTCTTC TTTCTCCAGG CTAGAAGAAC

AAAAGAATAT CTTGTCAGAA TTTCAAAGAG ATTTAAATGA ATTTGTTTTA

TGGTTGGAGG AAGCAGATAA CATTGCTAGT ATCCCACTTG AACCTGGAAA

AGAGCAGCAA CTAAAAGAAA AGCTTGAGCA AGTCAAGGTA ATTTTATTTT

CTCAAATCCC CCAGGGCCTG CTTGCATAAA GAAGTATATG AATCTATTTT

TTAATTCAAT CATTGGTTTT CTGCCCATTA GGTTATTCAT AGTTCCTTGC

TAAAGTGTTT TTCTCACAAC TTTATTTCTT CTTAACCCTG CAGTTCTGAA

CCAGTGCACA TAAGAACATA TGTATATATG TGTGTGTGTG TATTTATATA

TACACACACA CATATTGCAT CTATACATCT ACACATATAG ATGTATAGAT

TCAATATGTC TAAAAATGTA TATAATTCAC AGTTTTATC TTTGATTTGA

ATATTTAAGG GACTGAGACT CACACTCATA TACTTTT-3' and fragments thereof, said fragments complementary to the sense and anti-sense strands of the gene coding for dystrophin, said fragments capable of annealing to said strands of the dystrophin gene and amplifying dystrophin sequences.