Process for analyzing length polymorphisms in DNA regions

Abstract

A process for analyzing length polymorphism in DNA regions wherein the following steps are carried out: (a) annealing at least one primer pair to the DNA to be analyzed, wherein one of the molecules of the primer pair is substantially complementary to one of the complementary strands of the 5′ or 3′ flank of a simple or cryptically simple DNA sequence, and wherein the annealing occurs in such an orientation that the synthesis products obtained by a primer-controlled polymerisation reaction with one of said primers can serve as template for annealing the other primer after denaturation; (b) primer-controlled polymerase chain reaction; and (c) separating and analyzing the polymerase chain reaction products.

We claim:  
     
       1. A method for determining length polymorphisms in a simple or cryptically simple sequence in one or more DNA regions of one or more subjects, which comprises: 
       a) providing at least one DNA sample, comprising a template DNA having a nucleotide sequence that includes a simple or cryptically simple sequence comprising trinucleotide repeats, from at least one subject;  
       b) annealing at least one primer pair to the template DNA of each of said DNA samples, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 5′ side of said simple or cryptically simple DNA sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the 3′ side of said simple or cryptically simple DNA sequence; wherein said first and second primers each anneal to a single site in said template DNA and the sequence of the template DNA between the sites where said primers anneal is 50 to 500 nucleotides in length;  
       c) performing at least one primer-directed polymerase chain reaction upon said template DNA having said primers annealed thereto, so as to form at least one polymerase chain reaction product;  
       d) separating the products of each polymerase chain reaction according to their lengths; and  
       e) analyzing the lengths of the separated products to determine the length polymorphisms of the simple or cryptically simple sequences.  
     
     
       2. The method according to  claim 1 , wherein each simple or cryptically simple DNA sequence comprises at least one trinucleotide motif. 
     
     
       3. The method according to  claim 1 , wherein each simple or cryptically simple DNA sequence contains  the trinucleotide repeat motif is CAG/CTG. 
     
     
       4. The method according to  claim 1 , wherein at least 2 primer pairs are used. 
     
     
       5. The method according to  claim 1 , wherein between 2 and 50 primer pairs are used. 
     
     
       6. The method according to  claim 1 , wherein each of the primers has a length ranging between 15 and 25 nucleotides. 
     
     
       7. The method according to claim  2   1 , wherein the annealing position of the primers of each pair is selected such that each of the primer-directed polymerase chain reaction products are separable one from the other as individual bands on a suitable electrophoretic gel. 
     
     
       8. The method according to  claim 1 , wherein the product of each primer-directed polymerase chain reaction is labelled by a radioactive label. 
     
     
       9. The method according to  claim 1 , wherein the product of each primer-directed polymerase chain reaction is labelled by a non-radioactive label. 
     
     
       10. The method according to  claim 9 , wherein said non-radioactive label is a fluorescent label. 
     
     
       11. The method according to  claim 1 , wherein said simple or cryptically simple DNA sequence is located adjacent to or within a genetically defined locus such that said simple or cryptically simple DNA sequence can serve as a marker for said locus. 
     
     
       12. A kit for performing the method of  claim 1 , comprising: 
       a) at least one vessel containing an equimolar mixture of primers constituting between 1 and 50 of said primer pairs;  
       b) a vessel containing a polymerizing enzyme suitable for performing a primer-directed polymerase chain reaction;  
       c) a vessel containing the deoxynucleotide triphosphates adenosine, guanine, cytosine and thymidine;  
       d) a vessel containing a buffer solution suitable for performing a polymerase chain reaction, or a concentrate of said buffer solution;  
       e) a vessel containing a template DNA that has a nucleotide sequence including a simple or cryptically simple sequence for assaying positive performance of the method, wherein each simple or cryptically simple DNA sequence comprises at least one trinucleotide motif.  
     
     
       13. The kit of  claim 12 , wherein at least one primer of each primer pair is labelled with a fluorescent or a radioactive label. 
     
     
       14. The method of  claim 1 , wherein the kinship of at least two subjects is determined by comparing length polymorphisms determined in step (e). 
     
     
       15. A method for analyzing length polymorphisms in at least one locus in an DNA sample obtained from at least one subject, wherein said DNA sample comprises a DNA template having at least one locus comprising a simple or cryptically simple DNA sequence, said method comprising: 
         a )  annealing said DNA template with at least one pair of primers, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking said simple or cryptically simple DNA sequence on the  5 ′ side of said simple or cryptically simple DNA sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the  3 ′ side of said simple or cryptically simple DNA sequence; wherein said first and second primers each anneal to a single site in said DNA template and wherein the annealing sites are separated by  50  to  500  nucleotides of template DNA;    
         b )  performing at least one primer - directed polymerase chain reaction upon said template DNA having said primers annealed thereto, so as to form at least one polymerase chain reaction product;    
         c )  separating the products of each polymerase chain reaction according to their lengths; and    
         d )  analyzing the lengths of the separated products to determine the length polymorphisms of said simple or cryptically simple sequences;    
       
         wherein said simple or cryptically simple sequence has a repeat length of  3  to  10  nucleotides.  
       
     
     
       16. The method of  claim 15 , wherein said simple or cryptically simple sequence has a repeat length of  3  to  6  nucleotides. 
     
     
       17. The method of  claim 15 , wherein said simple or cryptically simple sequence has a repeat length of  3  nucleotides. 
     
     
       18. The method of  claim 15 , wherein at least two primer pairs are used. 
     
     
       19. The method of  claim 15 , wherein the kinship of at least two subjects is determined by comparing the length polymorphisms determined in step ( d ) .   
     
     
       20. The method of  claim 16 , wherein the kinship of at least two subjects is determined by comparing the length polymorphisms determined in step ( d ) .   
     
     
       21. The method of  claim 15 , further comprising ( e )  comparing the length polymorphisms determined in step  ( d )  to numerical values of length polymorphisms for said at least one locus.   
     
     
       22. The method of  claim 16 , further comprising ( e )  comparing the length polymorphisms determined in step  ( d )  to numerical values of length polymorphisms for said at least one locus.   
     
     
       23. The method of  claim 15 , wherein  2  to  50  primer pairs are used. 
     
     
       24. The method according to  claim 15 , wherein each of the primers has a length of  15  to  25  nucleotides. 
     
     
       25. The method according to  claim 15 , wherein the annealing position of the primers of each pair is selected such that each of the products of the primer- directed polymerase chain reaction is separable one from the other as individual bands on a suitable electrophoretic gel.   
     
     
       26. The method according to  claim 15 , wherein the product of each primer- directed polymerase chain reaction is labeled by a radioactive label.   
     
     
       27. The method according to  claim 15 , wherein the product of each primer- directed polymerase chain reaction is labeled by a non - radioactive label.   
     
     
       28. The method according to  claim 27 , wherein said non- radioactive label is a fluorescent label.   
     
     
       29. The method according to  claim 15 , wherein said simple or cryptically simple DNA sequence is located adjacent to or within a genetically defined locus such that said simple or cryptically simple DNA sequence can serve as a marker for said locus. 
     
     
       30. The method of  claim 15 , wherein said simple or cryptically simple sequence has a repeat length of  3  to  6  nucleotides, each of said primers has a length of  15  to  25  nucleotides and the kinship of at least two subjects is determined by comparing the length polymorphisms determined in step ( d ) .   
     
     
       31. The method of  claim 15 , wherein said simple or cryptically simple sequence has a repeat length of  3  to  6  nucleotides, each of said primers has a length of  15  to  25  nucleotides and wherein  2  to  50  primer pairs are used. 
     
     
       32. The method of  claim 29 , wherein said simple or cryptically simple sequence has a repeat length of  3  to  6  nucleotides, each of said primers has a length of  15  to  25  nucleotides and  2  to  50  primer pairs are used. 
     
     
       33. The method of  claim 30 , wherein said simple or cryptically simple sequence has a repeat length of 3  nucleotides.   
     
     
       34. The method of  claim 32 , wherein said simple or cryptically simple sequence has a repeat length of  3  nucleotides. 
     
     
       35. A kit for performing analysis of polymorphism in simple or cryptically simple sequences, comprising: 
         a )  at least one vessel containing a mixture of primers constituting  1  to  50  primer parts; wherein each of said primer pairs is composed of a first primer complementary to a nucleotide sequence flanking said simple or cryptically simple DNA sequence on the  5 ′ side of said simple or cryptically simple DNA sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the  3 ′ side of said simple or cryptically simple DNA sequence; wherein said first and second primers each anneal to a single site in said DNA template and wherein the annealing sites are separated by  50  to  500  nucleotides of template DNA;    
         b )  a vessel containing a template DNA that has a nucleotide sequence including a simple or cryptically simple sequence for assaying positive performance of the method.   
     
     
       36. The kit of  claim 35 , wherein at least one primer of each primer pair is labeled with a fluorescent or a radioactive label. 
     
     
       37. The kit of  claim 35 , wherein said simple or cryptically simple sequence has a repeat length of  3  to  6  nucleotides and each of said primers has a length of  15  to  25  nucleotides. 
     
     
       38. The kit of  claim 35 , wherein said simple or cryptically simple DNA sequence is located adjacent to or within a genetically defined locus such that said simple or cryptically simple DNA sequence can serve as a marker for said locus. 
     
     
       39. The kit of  claim 38 , wherein at least one primer of each primer pair is labeled with a fluorescent or a radioactive label. 
     
     
       40. A method for determining length polymorphisms in a simple or cryptically simple sequence in one or more DNA regions of one or more subjects, which comprises: 
         a )  providing at least one DNA sample, comprising a template DNA consisting essentially of a nucleotide sequence that includes i )  a simple or cryptically simple sequence having a trinucleotide repeat motif and ii )  nucleotide sequences flanking the simple or cryptically simple sequence, from at least one subject;    
         b )  annealing at least one primer pair to the template DNA of each of said DNA samples, wherein said primer pair is composed of a first primer complementary to the nucleotide sequence flanking the simple or cryptically simple DNA sequence on the  5 ′ side of said simple or cryptically simple DNA sequence and a second primer complementary to the nucleotide sequence flanking the simple or cryptically simple DNA sequence on the  3 ′ side of said simple or cryptically simple DNA sequence; wherein said first and second primers each anneal to a single site in said template DNA and the sequence of the template DNA between the sites where said primers anneal is  50  to  500  nucleotides in length;    
         c )  performing at least one primer - directed polymerase chain reaction upon said template DNA having said primers annealed thereto, so as to form at least one polymerase chain reaction product;    
         d )  separating the products of each polymerase chain reaction according to their lengths;    
         e )  analyzing the lengths of the separated products to determine the length polymorphisms of the simple or cryptically simple sequences.   
     
     
       41. A method for analyzing polymorphism in at least one locus in an DNA sample comprising a DNA template, said method comprising: 
         a )  annealing said DNA template with at least one pair of primers, wherein said primer pair is composed of a first primer complementary to a nucleotide sequence flanking said simple or cryptically simple DNA sequence on the  5 ′ side of said simple or cryptically simple DNA sequence and a second primer complementary to a nucleotide sequence flanking the simple or cryptically simple DNA sequence on the  3 ′ side of said simple or cryptically simple DNA sequence; wherein said first and second primers each anneal to a single site in said DNA template and wherein the annealing sites are separated by  50  to  500  nucleotides of template DNA;    
         b )  performing at least one primer - directed polymerase chain reaction upon said template DNA having said primers annealed thereto, so as to form at least one polymerase chain reaction product;    
         c )  separating the products of each polymerase chain reaction product according to their lengths; and    
         d )  analyzing the lengths of the separated products to determine the length polymorphisms of said simple or cryptically simple sequences,    
       
         wherein said DNA template includes at least one sequence consisting essentially of a simple or cryptically simple DNA sequence having a repeat motif length of  3  to  10  nucleotides and nucleotide sequences flanking said simple or cryptically simple DNA sequence effective for annealing said at least one pair of primers. 
       
     
     
       42. A kit for analyzing polymorphism in at least one locus in an DNA sample, comprising: 
         a )  at least one vessel containing a mixture of primers constituting between  1  and  50  of said primer pairs;    
         b )  a vessel containing a polymerizing enzyme suitable for performing a primer - directed polymerase chain reaction;    
         c )  a vessel containing the deoxynucleotide triphosphates adenosine, guanine, cytosine and thymidine;    
         d )  a vessel containing a buffer solution for performing a polymerase chain reaction;    
         e )  a vessel containing a template DNA comprising i )  a simple or cryptically simple nucleotide sequence having a repeat motif length of  3  to  10  nucleotides and ii )  nucleotide sequences flanking said simple or cryptically simple nucleotide sequence that are effective for annealing at least one pair of said primers, for assaying positive performance of the method.