WO2001040508A1

WO2001040508A1 - Unique adaptor design for amplified fragment length polymorphism (aflp) fingerprinting

Info

Publication number: WO2001040508A1
Application number: PCT/AU2000/001484
Authority: WO
Inventors: Peter Richard Reeves
Original assignee: University of Sydney
Current assignee: University of Sydney
Priority date: 1999-12-01
Filing date: 2000-12-01
Publication date: 2001-06-07
Anticipated expiration: 2002-06-01
Also published as: AUPQ439699A0

Abstract

The invention relates to improved Amplified Fragment Length (AFLP) DNA fingerprinting based upon the design of unique adaptor molecules.

Description

Unique Adaptor Design for Amplified Fragment Length Polymorphism (AFLP) Fingerprinting

Technical Field of the Invention

The invention relates to novel nucleic acid molecules and the use of those molecules for the detection of nucleic acid fragments.

Background of the Invention

The analysis of genetic polymorphisms at the level of nucleotide sequences typically involves the display of a set of nucleic acid fragments from a specific genomic sample generated under a specific set of conditions and the comparison of those fragments with a set of nucleic acid fragments derived from another genomic sample under the same conditions. These analyses find application in inter alia, the determination of the genetic similarity of biological samples, in particular forensic samples, microbial, plant or animal samples and in the determination of pedigree. A genetic polymorphism can be detected by the

Amplified Fragment Length Polymorphism (AFLP) technique. In the first step of AFLP, DNA is digested with restriction enzymes, in particular, a frequent cutter enzyme which has a recognition sequence of 4 nucleotides, and an infrequent cutter enzyme which has a recognition sequence of 6 nucleotides. The digestion produces three subsets of digested fragments: those fragments with 'like ends', which are (i) fragments flanked by an overhang sequence created by digestion with the frequent cutter (the 'frequent cutter overhang' ) and (ii) fragments flanked by an overhang sequence created by digestion with the infrequent cutter (the 'infrequent cutter overhang'); and (iii) those fragments with ^"-hetero ends', which are fragments which have a frequent cutter overhang at one end of the fragment a.id an infrequent cutter overhang at the opposite end of the fragment .

In the second step of AFLP, double stranded adaptors that comprise a core nucleotide sequence that is flanked on one end by an infrequent or frequent cutter overhang are ligated to the ends of the fragments so that the overhang of the adaptors hybridise with the overhang of the fragments. The adaptors are not phosphorylated at the terminal 5' nucleotide so that the formation of a phosphodiester bond is limited to the 5' end of the fragment and the 3' end of the adaptor molecule. The ligation produces three sub-sets of ligation products: (i) a fragment ligated to adaptors via frequent cutter overhangs; (ii) a fragment ligated to adaptors via infrequent cutter overhangs and (iii) a fragment ligated to an adaptor via a frequent cutter overhang at one end of the fragment, and ligated to an adaptor via an infrequent cutter overhang at the opposite end of the fragment. In the final step, the ligation products are amplified. The nucleotide sequences of the primers used in the amplification step comprise a sequence which is complementary to the core sequence of the adaptor, a sequence which is complementary to the infrequent or frequent cutter overhang of the adaptor and at least one (but typically three) 'selective nucleotide' at the 3' terminal region of the primer which is complementary to the sequence of the ligation product located 3' to the phosphodiester linkage created in the second step of the method. The selective nucleotide increases the specificity of primer annealing because each selective nucleotide restricts the fragments which can be amplified by the primer to those fragments which have nucleotides which are complementary to the selective nucleotides and are capable of hybridising to the selective nucleotide when the primer binds the core sequence of the adaptor and the infrequent or frequent cutter overhang of the adaptor. Thus the selective nucleotides are used to bring the number of amplified fragments within the range suitable for subsequent analysis of those fragments. After annealing of a pair of primers, synthesis commences from the 3 ' ends of the primers so that both strands of the ligation product are amplified.

A pre-amplification step which comprises amplification with a primer pair comprising only one selective nucleotide usually precedes the amplification step. The combination of frequent cutter and infrequent cutter enzymes is required in AFLP because the technique is based on detection of digested fragments by amplification of the digested fragments. To be able to detect the digested fragments, the number of the digested fragments must be sufficient to allow amplification. The digested fragments must also be of a size which is sufficient to allow the digested fragments to be amplified, and sufficient to allow the amplification product to be identified, for example by gel electrophoresis .

Digested fragments which have hetero ends are of a number and size which is sufficient to allow detection by amplification of those fragments. The number and size of digested fragments with like ends is not sufficient to allow detection of those fragments by amplification.

Specifically, fragments digested by a frequent cutter only will be too numerous for detection by amplification, and fragments which are digested by an infrequent cutter only will be too large and often too few for detection by amplification.

As digested fragments with like ends and hetero ends are produced in AFLP, it is essential that the amplification of the digested fragments be biased in favour of amplification of fragments with hetero ends rather than like ends. By keeping amplification of digested fragments with like ends to a minimum, a signal to noise ratio is achieved which is sufficient to allow detection of fragments with hetero ends by amplification. In use, however, a serious limitation applies to the sensitivity of AFLP because the vast majority of the digested fragments (>90%) are fragments with 'like ends' digested by the frequent cutter. This means that it is difficult to achieve a signal to noise ratio which is sufficient to allow the detection of digested fragments with hetero ends .

Summary of the Invention The invention seeks to minimise the above-mentioned limitation by providing adaptor molecules and a method of using the adaptor molecules for the detection of nucleic acid fragments.

In one aspect the invention provides a first adaptor molecule comprising first and second strands which are hybridised to each other so that an extension sequence of the second strand extends from the nucleotide of the second strand which is hybridised with the 3' terminal nucleotide of the first strand, the extension sequence in use being capable of hybridising to a further strand, wherein the 3' terminal nucleotide of the first strand is arranged so that the 3' terminal nucleotide is not capable of forming a phosphodiester bond with a nucleotide and wherein the 5' terminal nucleotide of the extension sequence is arranged to allow the 5' terminal nucleotide to form a phosphodiester bond with a nucleotide.

As described further herein, the arrangement of the 3' terminal nucleotide of the first strand prevents exponential amplification of digested fragments which have like ends generated by an infrequent cutter enzyme. It will be understood that there are a number of different arrangements of the 3' terminal nucleotide which may be made so that the 3' terminal nucleotide is not capable of forming a phosphodiester bond with a nucleotide. In one embodiment, the 3' terminal nucleotide may be directly modified so as to produce a structure which is not capable of forming a phosphodiester bond with a nucleotide. For example, the 3' terminal nucleotide may be a di- deoxyribonucleotide . In another embodiment, the 3' terminal nucleotide may be chemically linked to a chemical group which prevents the 3' terminal nucleotide forming a phosphodiester bond with a nucleotide. For example, the 3' terminal nucleotide may be linked to an amino group which blocks the 3 ' hydroxyl group of the 3 ' terminal nucleotide forming a phosphodiester bond with a nucleotide .

In one embodiment, the 5' terminal nucleotide of the extension sequence of the second strand is arranged to allow the 5' terminal nucleotide to form a phosphodiester bond with a further nucleotide, by phosphorylation of the 5' terminal nucleotide. As described above, adaptor molecules used in AFLP before the invention are not phosphorylated, so as to prevent the adaptor molecules ligating during the ligation step. Advantageously, the adaptation of the 3' terminal nucleotide of the first strand prevents the first adaptor molecules ligating with each other, and this allows the 5' terminal nucleotide of the extension sequence of the second strand to be arranged to allow the 5' terminal nucleotide to form a phosphodiester bond with a nucleotide, for example by phosphorylation of the 5' terminal nucleotide.

As described further herein, the arrangement of the 5' terminal nucleotide of the extension sequence of the second strand to form a phosphodiester bond allows a 'hot start' step to be incorporated when using the adaptor molecules. The hot start step increases the specificity of the amplification procedure. As the adaptor molecules used in AFLP before the invention are not phosphorylated, a hot start step cannot be incorporated when using these adaptor molecules. The nucleotide sequence of the extension sequence of the second strand is complementary to an overhang of a digested fragment which is generated when a double stranded nucleic acid molecule has been digested by a restriction endonuclease . In one embodiment, the nucleotide sequence of the extension sequence is complementary to an overhang of a digested fragment which is generated when a double stranded nucleic acid molecule has been digested by an infrequent cutter enzyme. Preferably the infrequent cutter enzyme is EcoRI . The extension sequence of the second strand has a length which is sufficient to allow the 5' terminal nucleotide of the extension sequence of the second strand to form a phosphodiester bond with the 3' terminal nucleotide at the recessed end of the digested fragment . For example, when the digested fragment is generated by digestion of a double stranded nucleic acid molecule with EcoRI, the extension sequence consists of four nucleotides (5' -TTAA-3' ) . In one embodiment, the 3' terminal nucleotide of the first strand prevents the first adaptor molecule from being cleaved from a digested fragment after the adaptor molecule has been ligated to the digested fragment. For example, when the extension sequence of the second strand is complementary to an overhang of a digested fragment which is generated when a double stranded nucleic acid molecule is cleaved with EcoRI, the 3' terminal nucleotide is preferably cytosine (or thymine or adenine) rather than guanine .

In one embodiment, the first strand has a shorter length than the second strand so that the second strand comprises a second extension sequence which extends from the nucleotide of the second strand which is hybridised to the 5' terminal nucleotide of the first strand. As described further herein, the second extension sequence allows for the pre- amplification of ligated products using a pre-amplification primer in the method of the invention described herein, regardless of the combination of frequent and infrequent cutter enzymes that are used.

In one embodiment, the second strand, including the second extension sequence is between 15 and about 35 nucleotides in length.

In one embodiment, the adaptor molecule of the first aspect of the invention is according to the adaptor molecule shown in Figure 1.

In a second aspect the invention provides a second adaptor molecule comprising first and second strands which are hybridised to each other so that an extension sequence of the second strand extends from the nucleotide of the second strand which is hybridised with the 3' terminal nucleotide of the first strand, the extension sequence in use being capable of hybridising to a further strand, wherein the 5' terminal nucleotide of the extension sequence of the second strand is arranged so that the 5' terminal nucleotide is not capable of forming a phosphodiester bond with a nucleotide. As described further herein, the arrangement of the 5' terminal nucleotide of the extension sequence of the second strand prevents amplification of digested fragments which have like ends generated by a frequent cutter enzyme. It will be understood that there are a number of different arrangements of the 5' terminal nucleotide which may be made so that the 5' terminal nucleotide is not capable of forming a phosphodiester bond with a nucleotide. In one embodiment, the 5' terminal nucleotide is not phosphorylated so that the 5' terminal nucleotide is not capable of forming a phosphodiester bond with a nucleotide. Advantageously, the adaptation of the 5' terminal nucleotide of the second strand prevents the second adaptor molecules ligating with each other.

The nucleotide sequence of the extension sequence of the second strand of the second adaptor molecule is complementary to an overhang of a digested fragment which is generated when a double stranded nucleic acid molecule has been digested by a restriction endonuclease . In one embodiment, the nucleotide sequence of the extension sequence of the second strand is complementary to an overhang of a digested fragment which is generated when a double stranded nucleic acid molecule has been digested by a frequent cutter enzyme. Preferably the frequent cutter enzyme is Msel . The extension sequence of the second strand of the second adaptor molecule has a length which is sufficient to allow the 3' terminal nucleotide of the first strand of the adaptor molecule to form a phosphodiester bond with the 5' terminal nucleotide of a digested fragment. For example, when the digested fragment is generated by digestion of a double stranded nucleic acid molecule with Msel, the extension sequence of the second strand of the adaptor consists of two nucleotides (5'-TA-3').

In one embodiment, the 3' terminal nucleotide of the first strand prevents the second adaptor molecule from being cleaved from a digested fragment after the adaptor molecule has been ligated to the digested fragment. For example, when the extension sequence is complementary to an overhang of a digested fragment which is generated when a double stranded nucleic acid molecule is cleaved with Msel, the 3' terminal nucleotide is preferably guanine (or thymine or cytosine) rather than adenine. In one embodiment, the 3' terminal nucleotide of the second strand of the second adaptor is arranged so as to prevent a primer from hybridising to the second strand. It will be understood that in some circumstances, particularly when a hot start is not used in the method of the invention described herein, the polymerase may cause the 5' terminal nucleotide of the extension sequence of the second strand to form a phosphodiester bond. When the 5' terminal nucleotide of the extension sequence is phosphodiester bonded, the arrangement of the 3' terminal nucleotide of the second strand prevents a primer from hybridising to the second strand when the adaptor molecule is used in the method of the invention described herein. The 3' terminal nucleotide of the second strand may be arranged by any modification which is capable of preventing a primer hybridising to the second strand. For example, the 3' terminal nucleotide may be chemically linked to an amino group so as to prevent a primer from hybridising to the second strand. In one embodiment, the second strand of the second adaptor has a shorter length than the first strand of the adaptor molecule, so that the first strand of the adaptor molecule comprises a second extension sequence which extends from the nucleotide of the first strand which is hybridised to the 3' terminal nucleotide of the second strand. As described further herein, the second extension sequence allows for the pre-amplification of ligated products using a pre-amplification primer in the method of the invention described herein, regardless of the combination of frequent and infrequent cutter enzymes that are used. In one embodiment, the first strand, including the second extension sequence is between 15 and about 35 nucleotides in length. In one embodiment, the adaptor molecule of the second aspect of the invention is according to the adaptor molecule shown in Figure 2.

In a third aspect, the invention provides a method for the detection of a nucleic acid molecule which has hetero ends in a sample of nucleic acid molecules, the method comprising the following steps: a) contacting a nucleic acid molecule in the sample which has an infrequent cutter overhang with a first adaptor molecule so that the extension sequence of the second strand of the first adaptor molecule hybridises with the infrequent cutter overhang and the 5' terminal nucleotide of the extension sequence of the second strand forms a phosphodiester bond with a 3' terminal nucleotide of the infrequent cutter overhang; b) contacting a nucleic acid molecule in the sample which has a frequent cutter overhang with a second adaptor molecule so that the extension sequence of the second strand of the second adaptor molecule hybridises with the frequent cutter overhang and the 3' terminal nucleotide of the first strand forms a phosphodiester bond with a 5' terminal nucleotide of the frequent cutter overhang; c) synthesising a third strand from the 3' end of a first primer which is hybridised to the second strand of the first adaptor so that the 3' end of the third strand comprises a nucleotide sequence which is complementary to the nucleotide sequence of the first strand of the second adaptor molecule; d) detecting the presence of the third strand.

The result of the formation of the phosphodiester bonds in steps a) and b) is that a single stranded nucleic acid molecule is formed which consists of the second strand of the first adaptor molecule phosphodiester bonded via the 5' terminal nucleotide of the extension sequence of the second strand to the 3' end of a single strand of a nucleic acid molecule in the sample, the 5' end of the nucleic acid molecule being phosphodiester bonded to the 3' terminal nucleotide of the first strand of the second adaptor molecule. This single stranded nucleic acid molecule is a template for the synthesis of the third strand from the first primer.

As the nucleotide sequence of the first primer is complementary to at least part of the second strand of the first adaptor molecule, optionally including the extension sequence of the second strand and the nucleotide which is phosphodiester bonded to the 5' terminal nucleotide of the extension sequence of the second strand, the third strand is synthesised only when the 5' terminal nucleotide of the extension sequence of the second strand of the first adaptor molecule is phosphodiester bonded to the nucleic acid molecule in the sample digested with an infrequent cutter. The detection of a synthesised third strand is therefore equivalent to the detection of a nucleic acid molecule in the sample which has an infrequent cutter overhang at one end of the nucleic acid molecule.

The sequence of the 3' end of the third strand is complementary to the sequence of the first strand of the second adaptor molecule if the 3' end of the first strand of the second adaptor molecule has formed a phosphodiester bond. Thus the detection of a third strand which comprises a nucleotide sequence which is complementary to the nucleotide sequence of the first strand of the second adaptor molecule at the 3' end of the third strand is equivalent to the detection of a nucleic acid molecule in the sample which has hetero ends (i.e. which has an infrequent cutter overhang at one end of the molecule and a frequent cutter overhang at the opposite end of the molecule) .

A third strand which comprises a nucleotide sequence which is complementary to the nucleotide sequence of the first strand of the second adaptor molecule at the 3' end of the third strand may be detected by synthesising a fourth strand from the 3' end of a second primer so that the 3' end of the fourth strand comprises a nucleotide sequence which is complementary to the nucleotide sequence of the first primer. Thus in one embodiment the method of the third aspect comprises the further steps of: e) synthesising a fourth strand from the 3' end of a second primer which is hybridised to the 3' end of the third strand so that the 3 ' end of the fourth strand comprises a nucleotide sequence which is complementary to the nucleotide sequence of the first primer and; f) detecting the presence of the fourth strand.

The effect of forming a single stranded template in steps a) and b) and the use of first and second primers for synthesis of the third and fourth strands is that a nucleic acid molecule in the sample with hetero ends can be exponentially amplified (Figure 3A) . A nucleic acid molecule which comprises a frequent cutter overhang at both ends cannot be amplified at all because the first primer is unable to hybridise to the first strand of the second adaptor molecule (Figure 3B) . A nucleic acid molecule which comprises an infrequent cutter overhang at both ends can be amplified, however, amplification is linear amplification because the 3' terminal nucleotide of the first strand of the first adaptor molecule is unable to form a phosphodiester bond, so that the second primer is unable to hybridise to the 3' end of the third strand (Figure 3C) . As there is likely to be a low number of nucleic acid molecules which have infrequent cutter overhangs at both ends, the level of amplification of these molecules is negligible. Thus the signal to noise ratio of the nucleic acid molecules which have hetero ends is increased by prevention, or otherwise suppression of the amplification of nucleic acid molecules which have like ends.

As described further herein, in certain circumstances, it may be necessary for the first primer to hybridise to a region of the second strand of the first adaptor molecule which includes the extension sequence of the second strand and the nucleotide which is phosphodiester bonded to the 5' terminal nucleotide of the extension sequence of the second strand. It may also be necessary for the second primer to hybridise to a region of the 3' end of the third strand which includes the sequence of the third strand which is complementary to the nucleotide located 3' adjacent to the nucleotide which is phosphodiester bonded to the 3' end of the first strand of the second adapter molecule. Such hybridisation of the first and second primers increases the specificity of the method of the invention for the detection of nucleic acid molecules which have hetero ends. This is because the 3' terminal nucleotides of these first and second primers limit the digested fragments which can be amplified to those fragments which have a nucleotide sequence which is capable of hybridising with the 3' terminal nucleotide of the first and second primers when the first and second primers hybridise to the second and third strands, respectively.

Thus in one embodiment of the method of the third aspect, in step c) , the 3' terminal nucleotide of the first primer is hybridised to the nucleotide which has formed a phosphodiester bond with the 5' terminal nucleotide of the extension sequence of the second strand of the first adaptor molecule. Preferably the first primer has the sequence shown in Figure 5. In another embodiment of the method of the third aspect, in step (e) the 3' terminal nucleotide of the second primer is hybridised to the nucleotide of the third strand which is complementary to a nucleotide located 3' adjacent to the nucleotide which is phosphodiester bonded to the 3' end of the first strand of the second adaptor molecule. Preferably the second primer has the nucleotide sequence shown in Figure 6.

Further, when analysing complex genomes, it may be necessary to use a first selective primer in step c) of the third aspect, in place of the first primer, for the synthesis of the third strand, the first selective primer being characterised in that the nucleotides at the 3' end of the primer are capable of hybridising to nucleotides which are 5' to the nucleotide which is phosphodiester bonded to the 5 'terminal nucleotide of the extension sequence of the second strand of the first adaptor. It may also be necessary to use a second selective primer in step e) of the third aspect, in place of the second primer, for the synthesis of the fourth strand, the second selective primer being characterised in that the nucleotides at the 3' end of the primer are capable of hybridising to nucleotides in a region of the 3' end of the third strand which is complementary to the nucleotides located 3' to the nucleotide which is phosphodiester bonded to the 3 ' end of the first strand of the second adaptor molecule.

Thus in another embodiment of the method of the third aspect, step c) comprises synthesising a third strand from the 3' end of a first selective primer, wherein the nucleotides at the 3' end of the first selective primer are capable of hybridising to nucleotides which are 5' to the nucleotide which is phosphodiester bonded to the 5' terminal nucleotide of the extension sequence of the second strand of the first adaptor molecule so that the 3' end of the third strand comprises a nucleotide sequence of the first strand of the second adaptor molecule. Preferably the first selective primer has a nucleotide sequence according to any one of the primers shown in Figure 7. In another embodiment of the method of the third aspect, step e) comprises synthesising a fourth strand from the 3' end of a second selective primer, wherein the nucleotides at the 3' end of the second selective primer are capable of hybridising to nucleotides in a region of the 3' end of the third strand which is complementary to the nucleotides located 3' to the nucleotide which is phosphodiester bonded to the 3' end of the first strand of the second adaptor molecule. Preferably the second selective primer has a nucleotide sequence according to any one of the primers shown in Figure 8.

As an alternative approach to the analysis of a complex genome, it may be necessary to pre-amplify the single stranded template produced after step b) of the method of the third aspect. In one embodiment, pre- amplification is caused by synthesising copies of the single stranded template from the 3' end of a first pre- amplification primer which hybridises to the second extension sequence of the second strand of the first adaptor molecule, and from the 3' end of a second pre- amplification primer which hybridises to the second extension sequence of the first strand of the second adaptor molecule. In one embodiment, the first and second pre-amplification primers have the nucleotide sequences shown in Figure 9.

As described above, an advantage of the adaptor molecules of the first and second aspect of the invention is that a 'hot start' step can be incorporated prior to the synthesis of the third strand. The 'hot start' step involves heating the sample of nucleic acid molecules prior to step c) to a temperature which prevents activity of a polymerase in the sample before the first primer is hybridised as in step c) . This increases the specificity of the method of the third aspect .

It will be understood that the method of the invention may include various combinations of the above described steps, including for example a hot start step prior to a pre-amplification, which is proceeded by amplification with the first and second primers, or alternatively, amplification with the first and second selective primers. Further, as described herein, where a sample of nucleic acid molecules which have been digested with a frequent and infrequent cutter enzyme, a digestion step may precede steps a) and b) of the method of the third aspect, or alternatively, the digestion may be performed at the same time as the ligation in steps a) and b) .

An amplified nucleic acid molecule which has hetero ends may be identified by electrophoresis in a suitable support, for example, polyacrylamide or agarose . In the circumstance where a complex mixture of amplified nucleic acid molecules with hetero ends require identification, it will be necessary to label the first or second primer, or the first or second selective primer, prior to the synthesis of the third or fourth strands. The capability to label the first or second primer, or the first or second selective primer is a significant advantage over AFLP methods before the invention. AFLP methods before the invention are limited to labelling the primer which hybridises to the adaptor molecule that is ligated to an infrequent cutter overhang on the digested fragment. This is because the ligation products in the AFLP methods before the invention are predominantly products which have like ends generated by a frequent cutter. With regard to the present invention, and as described further herein, labelling the second primer or the second selective primer produces a greater signal to noise ratio, as compared with labelling the first primer or the first selective primer, because in accordance with the method of the invention there is no amplification of a digestion fragment with like ends generated by a frequent cutter, and there is linear amplification of a digestion fragment generated by an infrequent cutter. It will be understood however, that as the amplification of digestion fragments with like ends generated by an infrequent cutter is negligible, the first primer or first selective primer may be labelled. The primers can be labelled with radiolabels including radiolabels such as ³²PγdATP, ³²PαdCTP, ³⁵SdATP. Alternatively, the primers can be labelled with fluorescent dyes such as fluoroscein or rhodamine .

Thus in one embodiment, one of the first or second primers, or first or second selective primers are labelled. Preferably the second primer or the second selective primer is labelled.

In a fourth aspect, the invention provides a method of detecting a polymorphism in a genome, the method according to the third aspect of the invention. In a fifth aspect the invention provides a kit comprising an adaptor molecule of the first and second aspects of the invention. In one embodiment, the kit comprises primers selected from the group consisting of first and second primers, first and second selective primers and pre-amplification primers, which are capable of hybridising with the adaptor molecules of the first and second aspects of the invention.

Brief Description of the Drawings Figure 1 shows the structure of a preferred first adaptor molecule.

Figure 2 shows the structure of a preferred second adaptor molecule .

Figure 3 represents the ligation of the first and second adaptor molecules shown in Figures 1 and 2 with (A) a nucleic acid molecule with hetero ends; (B) a nucleic acid molecule flanked by infrequent cutter overhangs; and (C) a nucleic acid molecule flanked by frequent cutter overhangs .

Figure 4 shows the nucleotide sequences of the first and second strands of the first and second adaptor molecules.

Figure 5 shows the nucleotide sequence of a preferred first primer of the invention.

Figure 6 show the nucleotide sequence of a preferred second primer of the invention. Figure 7 shows the nucleotide sequences of preferred first selective primers of the invention.

Figure 8 shows the nucleotide sequences of preferred second selective primers of the invention.

Figure 9 shows the nucleotide sequences of preferred pre-amplification primers of the invention.

Figure 10 shows AFLP patterns for K12 , yeast, and corn DNA. Lanes 1, 2, 7 and 8 (E. coli K-12), 3, 4, 9 and 10 (yeast) , and 5, 6, 11 and 12 (corn) were used in an AFLP. Pre-amplification was used in the method for lanes 1 to 6. Samples in lanes 1 to 6 are labelled with Eco RI primer. Samples in lanes 7 to 12 are labelled with Mse I primer. K12 was amplified with EcoRI+A and Msel+C primer combination, yeast with EcoRI +ACC and Msel+C, and corn with EcoRI+ACC and Msel+CTG.

Examples Figure 1 shows a preferred first adaptor molecule of the invention. The preferred first adaptor molecule comprises a first strand of 10 nucleotides in length which has an amino group attached to the 3' terminal nucleotide. There is a second strand of 35 nucleotides in length which is phosphorylated at the 5' terminal nucleotide of the second strand. Watson -Crick base pairing of the first and second strands causes the strands to hybridise so that an extension sequence of the second strand is provided which is complementary to the sequence of an infrequent cutter overhang generated by digestion with EcoRI. The length of the extension sequence of the second strand is sufficient to allow Watson-Crick base pairing between the extension sequence and an infrequent cutter overhang generated by digestion with Eco RI .

The phosphorylation of the 5' terminal nucleotide of the extension sequence of the second strand allows phosphodiester bonding between the 5' terminal nucleotide of the extension sequence of the second strand and the 3 ' terminal nucleotide of the recessed end of the infrequent cutter overhang generated by digestion with EcoRI.

The amino group which is bonded to the 3' terminal nucleotide of the first strand prevents phosphodiester bonding of the 3' terminal nucleotide with a phosphorylated 5' terminal nucleotide of the infrequent cutter overhang. The amino group also prevents self ligation of the adaptor.

The second extension sequence of the second strand of approximately 20 nucleotides in length is sufficient to allow a pre-amplification primer to hybridise at high stringency during a pre-amplification step.

Figure 2 shows a preferred second adaptor molecule of the invention. The preferred second adaptor molecule comprises a first strand of 33 nucleotides in length. There is a second nucleic acid strand of 12 nucleotides in length, the 3' terminal nucleotide being linked to an amino group, and 5' terminal nucleotide is non- phosphorylated. Watson-Crick base pairing of the first and second strands causes the first and second strands to hybridise so that an extension sequence of the second strand is provided which is complementary to the sequence of a frequent cutter overhang generated by digestion with Msel . The length of the extension sequence is sufficient to allow Watson-Crick base pairing between the extension sequence and a frequent cutter overhang generated by digestion with Mse I .

The non-phosphorylated 5' terminal nucleotide of the second strand prevents phosphodiester bonding between the 5' terminal nucleotide and 3' terminal nucleotide in the recessed end frequent cutter overhang which has been generated by digestion with Msel. It also prevents self ligation of the adaptor molecule.

The 3' terminal nucleotide of the first strand is capable of forming a phosphodiester bond with a 5' terminal nucleotide which forms a recessed end of a frequent cutter overhang which has been generated by digestion with Mse I.

The nucleotide sequence of the first strand of the second adaptor molecule is identical to the sequence of the primer which is used to initiate fourth strand synthesis. This prevents amplification of nucleic acid molecules which have like ends generated by digestion with a frequent cutter. The second extension sequence of the first strand of approximately 20 nucleotides in length is sufficient to allow a pre-amplification primer to hybridise at high stringency during a pre-amplification step. The amino group which is bonded to the 3' terminal nucleotide of the second strand prevents the second primer or second selective primer from binding to the second strand. METHODS

1. Preparation of adaptors

First adaptor molecule: #924/#925 Second adaptor molecule #930/#931

First adaptor Volume Second adaptor Volume molecule molcule

#924 (300pMol/ul) 20 930 (300pmol/ul) 20

#925 (300pM01/ul) 20 931 (300pMol/ul) 20

10xOPA+ 6 10XOPA+ 6

H₂0 74 H₂0 74

Total 120 Total 120

Prepare reaction solutions as described above, overlay with mineral oil and boil for 2 minutes cool to room temperature.

2. Digestion of nucleic acid and ligation or adaptors

Protocol 1: digestion and ligation.

Digestion

0.5ug DNA (lOOmg/mL for bacteria, 250mg/mL for yeast and

500mg/mL for mammalian genome) 5 U EcoRI 5 U Msel

8 μl OPA+ buffer (Pharmacia) 4 μl BSA lmg/ml H20 to 40μl

Incubate for 3 hrs at 37°C (ii) Ligation

Add lOμl to 40μl of digestion prepared at (i) .

First adaptor molecule lμl (of stock prepared above)

Second adaptor molecule lμl (of stock prepared above)

25mMATP lμl

OPA+ 2μl

BSA lmg/ml lμl

Ligase 1U

H0 to lOμl

Overlay mineral oil, incubate overnight at 37°C.

Protocol 2: concurrent digestion and ligation.

DNA 0.5ug lμl

EcoRI 5U-0.5μl

Msel 5U-0.5μl

OPA+ lOμl

T4 DNA Ligase IU-lμl of lU/μl first adaptor molecule lμl (of stock prepared above) second adaptor molecule lμl (of stock prepared above) lOMmatp lμl

BSA lμg/ul 5μl H₂0 29μl

Total 50μl

Overlay with mineral oil, leave the digestion and ligation at 37°C for at least 5 hours or overnight. Denature ligation at 94 °C for 2 minutes

PreAFLP (optional for small genomes)

Prepare a PCR cocktail . 1.

4mM dNTPs lμl

6pMol/ul 926 lμl 6pMol/ul 932 lμl lOx buffer II 2μl

Mg++(25 mM) 1.6μl

BSA 0.4μl

AmpliTaq Gold O.lμl H20 11.9μl

Total 19μl

Add lul DNA to 19ul of above cocktail

3. Perform PCR using the following cycles temp/cycles 1 20

94°C 10' 15'

56° 30'

72° 2'

4 If using Corbet machines, set ramping rate to

2 (l°C/2sec) . 5. Dilute PCR 1:10 and store at -20°C.

4. Label selective amplification primer

primer 3.3 (20pmoles) OPA+ 1 μl

T4 PNK 0.2 H₂0 3.5 Total 10

Incubate for 37°C for 2 hours, then add equal amount H₂0,

5. AFLP

1) Prepare a cocktail

dNTPs lμl

Selective Primer (labelled) 1 μl

Selective Primer 1 μl lOx buffer II 2 μl

Mg++ (25mM) 1.6 μl

BSA 0.4 μl

AmpliTaq Gold 0.1 μl

H₂0 11.9 μl

Total 19 μl

2) PCR

Add 1 μl of DNA (diluted preAFLP PCR or ligated chromosomal DNA) to 19ul of cocktail as above.

3) Cycles

Temperature/cycles 1 10+20* 1

94°C 10' 15"

66-56 °C ^# 30"

Touch down 1°C per cycle for 10 cycles from 65 to 56 °C. *If omit preAFLP step, increase 56 °C cycle to 25.

6 Gel electrophoresis

1) use 6% polyacrylamide gel

2) Prerun gel at 600V for 30 min

3) Mix 5μl of AFLP PCR product with 2.5μl loading dye, denature at 94°C

4) Run at 1200V for 3.5 hours 5) Transfer to 3MM filter paper

6) Vacuum dry at 80 °C for 1 hour

7) Expose to X-ray film for 24-48 hours

The method of the invention was tested using E. coli K-12, yeast, and corn DNA. 0.1 μg and 0.2 μg and 0.5 μg of DNA respectively were used for digestion and ligation of adaptors. 1 μl of the ligation mix is mixed with 19 μl of PCR mix containing 0.3 μm unlabelled primer and 0.1 μm ³³P labelled primer, 0.2 μm dNTPs, BSA (4 μg/ul), buffer (lOmM Tris-HCl, pH 8.3, 50 mM KC1 , and 2.5 mM MgCl₂) , and 0.5 unit AmpliTaq Gold (PE Applied Biosystems) . Basic cycling parameters are denaturation at 94 °C for 15s except for 10 min for the first cycle, annealing at temperatures specified below for 30 s, and extension at 72 °C for 1 min. The first 10 cycles were touch-down from 66 56°C decreasing by l°C/cycle and then 20 cycles at 56°C for preamplified samples and 25 cycles if without preamplification. Labelled amplification products were run on standard 6% polyacrylamide sequencing gels and visualised by exposure to Kodak BioMax-MR film overnight.

The AFLP fingerprinting patterns are shown in Fig. 10. We tested the effect of pre-amplification and the difference between labelling the first primer or second primer. Fingerprints for K12 are identical with and without preamplification when the second primer is labelled and similar but with a much higher background when the first primer is labelled. Some differences in patterns for yeast were seen with and without preamplification. There is a dramatic difference in patterns for the corn DNA indicating that selective amplification is unachievable without preamplification. The preamplification step for small genomes is not necessary, which is beneficial in diagnosis of bacterial pathogens or other situations where time is crucial .

With preamplification there is no difference between using labelled first primer or second primer. Both produced clear patterns with hardly any background. However without preamplification, labelling the second primer consistently gave clearer results than labelling the first primer, most likely because EcoRI -EcoRI fragments are linearly amplified which contributed to background. We recommend labelling the second primer as there is an additional benefit which is that only 6 base cutter needs to be changed to explore the genome space for other variation with the same frequent cutter, while both frequent and infrequent cutter need to be changed if the first primer is labelled. This may not be an issue for large genomes as there is a large number of selective primer combinations of three-base extension. But for bacterial genomes selective primers with one base extension are usually used and there are only 16 combinations of selective primer pairs from the four plus- one extensions for each of the first and second primers which may not generate sufficient differentiating fingerprints and require use of alternative restriction enzymes .

Claims

1. A first adaptor molecule comprising first and second strands which are hybridised to each other so that an extension sequence of the second strand extends from the nucleotide of the second strand which is hybridised with the 3' terminal nucleotide of the first strand, the extension sequence in use being capable of hybridising to a further strand, wherein the 3' terminal nucleotide of the first strand is arranged so that the 3' terminal nucleotide is not capable of forming a phosphodiester bond with a nucleotide and wherein the 5' terminal nucleotide of the extension sequence is arranged to allow the 5' terminal nucleotide to form a phosphodiester bond with a nucleotide .

2. A molecule according to claim 1 wherein the 3 ' terminal nucleotide of the first strand is a structure not capable of forming a phosphodiester bond with a nucleotide .

3. A molecule according to claim 2 wherein the structure is a di-deoxyribonucleotide .

4. A molecule according to claim 1 wherein the 3' terminal nucleotide of the first strand is chemically linked to a chemical group so as to be not capable of forming a phosphodiester bond with a nucleotide.

5. A molecule according to claim 4 wherein the chemical group is an amino group.

6. A molecule according to claim 1 wherein the 5' terminal nucleotide of the extension sequence is phosphorylated to allow the 5' terminal nucleotide to form a phosphodiester bond with a nucleotide.

7. A molecule according to claim 1 wherein the nucleotide sequence of the extension sequence is complementary to an overhang of a digested fragment which is generated when a double stranded nucleic acid molecule has been digested by an infrequent cutter enzyme.

8. A molecule according to claim 7 wherein the infrequent cutter enzyme is Eco RI .

9. A molecule according to claim 1 wherein the extension sequence consists of the sequence: 5'-TTAA-3'.

10. A molecule according to claim 1 wherein the 3' terminal nucleotide of the first strand prevents the first adaptor molecule from being cleaved from a digested fragment after the adaptor molecule has been ligated to the digested fragment.

11. A molecule according to claim 1 wherein the first strand has a shorter length than the second strand so that the second strand comprises a second extension sequence which extends from the nucleotide of the second strand which is hybridised to the 5' terminal nucleotide of the first strand.

12. A molecule according to claim 11 wherein the second strand is between 15 and about 35 nucleotides in length.

13. A first adaptor molecule as shown in Figure 1.

14. A second adaptor molecule comprising first and second strands which are hybridised to each other so that an extension sequence of the second strand extends from the nucleotide of the second strand which is hybridised with the 3' terminal nucleotide of the first strand, the extension sequence in use being capable of hybridising to a further strand, wherein the 5' terminal nucleotide of the extension sequence of the second strand is arranged so that the 5' terminal nucleotide is not capable of forming a phosphodiester bond with a nucleotide.

15. A molecule according to claim 14 wherein the 5' terminal nucleotide of the extension sequence is not phosphorylated so as to be not capable of forming a phosphodiester bond with a nucleotide.

16. A molecule according to claim 14 wherein the nucleotide sequence of the extension sequence of the second strand is complementary to an overhang of a digested fragment which is generated when a double stranded nucleic acid molecule has been digested by a frequent cutter enzyme.

17. A molecule according to claim 16 wherein the frequent cutter enzyme is Mse I.

18. A molecule according to claim 14 wherein the extension sequence consists of the sequence: (5'-TA-3').

19. A molecule according to claim 14 wherein the 3' terminal nucleotide of the first strand prevents the second adaptor molecule from being cleaved from a digested fragment after the adaptor molecule has been ligated to the digested fragment .

20. A molecule according to claim 14 wherein the 3' terminal nucleotide of the second strand is arranged so as to prevent a primer from hybridising to the second strand.

21. A molecule according to claim 20 wherein the 3' terminal nucleotide of the second strand is chemically linked to a chemical group so as to prevent a primer from hybridising to the second strand.

22. A molecule according to claim 21 wherein the chemical group is an amino group.

23. A molecule according to claim 14 wherein the second strand has a shorter length than the first strand so that the first strand comprises a second extension sequence which extends from the nucleotide of the first strand which is hybridised to the 3' terminal nucleotide of the second strand.

24. A molecule according to claim 14 wherein the first strand is between 15 and about 35 nucleotides in length.

25. A second adaptor molecule as shown in Figure 2

26. A method for the detection of a nucleic acid molecule which has hetero ends in a sample of nucleic acid molecules, the method comprising the following steps: a) contacting a nucleic acid molecule in the sample which has an infrequent cutter overhang with a first adaptor molecule according to claim 1 so that the extension sequence of the second strand of the first adaptor molecule hybridises with the infrequent cutter overhang and the 5' terminal nucleotide of the extension sequence of the second strand of the first adaptor molecule forms a phosphodiester bond with a 3' terminal nucleotide of the infrequent cutter overhang; b) contacting a nucleic acid molecule in the sample which has a frequent cutter overhang with a second adaptor molecule according to claim 14 so that the extension sequence of the second strand of the second adaptor molecule hybridises with the frequent cutter overhang and the 3 ' terminal nucleotide of the first strand of the second adaptor molecule forms a phosphodiester bond with a 5' terminal nucleotide of the frequent cutter overhang; c) synthesising a third strand from the 3' end of a first primer which is hybridised to the second strand of the first adaptor molecule so that the 3' end of the third strand comprises a nucleotide sequence which is complementary to the nucleotide sequence of the first strand of the second adaptor molecule; d) detecting the presence of the third strand, to detect a nucleic acid molecule which has hetero ends in the sample.

27. A method according to claim 26, further comprising: e) synthesising a fourth strand from the 3' end of a second primer which is hybridised to the 3' end of the third strand so that the 3 ' end of the fourth strand comprises a nucleotide sequence which is complementary to the nucleotide sequence of the first primer and; f) detecting the presence of the fourth strand, to detect a nucleic acid molecule which has hetero ends in the sample.

28. A method according to claim 26, wherein in step c) , the 3' terminal nucleotide of the first primer is hybridised to the nucleotide which has formed a phosphodiester bond with the 5' terminal nucleotide of the extension sequence of the second strand of the first adaptor molecule.

29. A method according to claim 28 wherein the first primer has the sequence shown in Figure 5.

30. A method according to claim 27 wherein in step e) the 3' terminal nucleotide of the second primer is hybridised to the nucleotide of the third strand which is complementary to a nucleotide located 3' adjacent to the nucleotide which is phosphodiester bonded to the 3' end of the first strand of the second adaptor molecule.

31. A method according to claim 30 wherein the second primer has the nucleotide sequence shown in Figure 6.

32. A method according to claim 26 wherein step c) comprises synthesising a third strand from the 3' end of a first selective primer, wherein the nucleotides at the 3' end of the first selective primer are capable of hybridising to nucleotides which are 5' to the nucleotide which is phosphodiester bonded to the 5' terminal nucleotide of the extension sequence of the second strand of the first adaptor molecule, so that the 3' end of the third strand comprises a nucleotide sequence of the first strand of the second adaptor molecule.

33. A method according to claim 32 wherein the first selective primer has a nucleotide sequence according to any one of the primers shown in Figure 7.

34. A method according to claim 27 wherein step e) comprises synthesising a fourth strand from the 3' end of a second selective primer, wherein the nucleotides at the 3' end of the second selective primer are capable of hybridising to nucleotides in a region of the 3' end of the third strand which is complementary to the nucleotides located 3' to the nucleotide which is phosphodiester bonded to the 3 ' end of the first strand of the second adaptor molecule.

35. A method according to claim 34 wherein the second selective primer has a nucleotide sequence according to any one of the primers shown in Figure 8.

36. A method according to claim 26 wherein prior to step c) pre-amplification strands are synthesised from the 3' end of a first pre-amplification primer which hybridises to the second extension sequence of the second strand of the first adaptor molecule and from the 3' end of a second pre-amplification primer which hybridises to the second extension sequence of the first strand of the second adaptor molecule.

37. A method according to claim 36 wherein the first and second pre-amplification primers have the nucleotide sequences shown in Figure 9.

38. A method according to any one of claims 26 to 37 wherein the first and/or second primers are labelled sufficient to allow detection of a nucleic acid molecule in the sample which has hetero ends.

39. A method according to any one of claims 26 to 37 wherein the second primer only, or the second selective primer only, is labelled.

40. Use of a method according to any one of claims 26 to 39 for detecting a polymorphism in a genome.

41. A kit comprising a first adaptor molecule according to claim 1 and second adaptor molecule according to claim 14.

42. A kit according to claim 41, further comprising primers selected from the group consisting of first and second primers, first and second selective primers and first and second pre-amplification primers.