In vitro method for providing templates for DNA sequencing

This application is the national phase under 35 U.S.C. §371 of PCT International Application No. PCT/F198/00586 which has an International filing date of Jul. 10, 1998 which designated the United States of America.

The present invention relates to the sequencing of DNA and in particular to the selection of unique templates for DNA sequencing. The selection methods of this invention can be performed fully in vitro. The invention further relates to a kit which can be used when employing the selection method of this invention.

DNA sequencing is a widely used method which is indispensable within modem molecular biology. One factor limiting sequencing is the shortness of the sequence to be read using one primer. Present-day techniques make it possible to read about 1000 bases starting from the primer. In practice, the range to be read covers about 400 to 800 bases. When sequencing a long DNA the examined DNA has been divided up into smaller fragments and the fragments have been separately sequenced, or the so called “DNA walk” method has been used.

In addition, it is essential that only one template is used in a single sequencing reaction. In the known methods, the problem has been solved by transforming the sequencing vector comprising the examined DNA into a bacterial cell, usually into Escherichia coli , and by isolating single colonies from the culture plate.

1. In the “DNA walk” method, the examined DNA is inserted into a vector and the inserted DNA is sequenced as far as possible by a known primer located in the vector. Using the obtained sequence, a new primer is designed, on which the sequencing is further continued (Itakura et al 1984 and Sambrook et al, 1989). The method is repeated until the entire sequence under examination has been exposed. The method is time-consuming and costly. The result from the previous round is needed for the next round, and new unique primers are required for each round.

2. The dividing up of the DNA into smaller, overlapping fragments has been performed by a number of different methods:

DNA mapping using restriction enzymes and subcloning of the restriction products into sequencing vectors (Sambrook et al, 1989);

the so called “shot gun” sequencing method where the DNA is randomly divided up into fragments and cloned into sequencing vectors (Sulston et al, 1992), and

the so called exodeletion method where the DNA under examination is ligated into a sequencing vector and shortened by means of an exonuclease (Sambrook et al, 1989 and Ausubel et al, 1989).

All of the above methods are laborious and in each one, the DNA must be transformed into a bacterium (usually into E. coli ) so as to produce a unique DNA template to be sequenced.

Furthermore, various kinds of methods based on DNA transposition have been disclosed for sequencing DNA. The majority of these methods are based on in vivo transposition (U.S. Pat. No. 4,716,105 and Strathmann et al 1991). The only known sequencing method (U.S. Pat. No. 5,728,551) exploiting in vitro transposition is based on using Ty1 integrase activity and even here, only the transposition reaction is carried out in vitro; the transposition products are transformed into Escherichia coli so as to obtain single sequencing templates. Related art is disclosed also in the U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,212,080, U.S. Pat. No. 5,645,991, and in Kasai et al (1992), Krishnan et al (1993), and Roach et al (1995).

It would be highly advantageous if the selection of single sequencing templates through a single cell culture step could be fully avoided. Even in the known, partly in vitro methods, the amplification products should be transformed into a bacterium and single colonies are to be picked in order to obtain unique templates for sequencing the DNA.

The present invention aims at eliminating the problems relating to the prior art and, therefore, it is an object of the present invention to provide a simple method for fragmenting larger DNA segments into pieces enabling the sequencing of whole segment by using one or a few sequencing primers.

It is another object of this invention to provide, fully in vitro, a unique sequencing template for a single DNA sequencing reaction.

According to one embodiment (later called embodiments A and B) of this invention the examined DNA is subjected to a DNA transposition reaction and to an amplification reaction. The amplification reaction is carried out in the presence of a first, fixed primer, hybridizing at a known position in the examined DNA or if the examined DNA is part of a vector for example, adjacent to the examined DNA, and a second, selective primer, hybridizing at the insertion site of a transposon. These reactions result in amplification products which can be separated on the basis of their size difference. By suitably selecting the amplification products, whose size difference is in the range of the reading capacity of the sequencing system used, normally in the range of 200 to 600 bp, overlapping DNA templates can be obtained for sequencing. The target DNA can range from a few base pairs up to 40 kilo base pairs. The only limiting factor for not choosing as target DNA even longer DNA segments is the inability of the present amplification reactions, such as PCR, to produce longer DNA segments.

According to another embodiment (later called embodiment C) of this invention a transposition reaction is carried out in the presence of the examined DNA and in the presence of a transposon or transposons which have different ends enabling the design of selective primers for both ends. The transposon ends can be part of the same molecule or separate molecules. The amplification reaction is carried out in the presence of the selective primers designed for both transposon ends. The amplification products are situated at random positions in the target DNA. To be able to cover the whole sequence of the target DNA, a sufficient number of the amplification products needs to be selected for sequencing. Very long DNA segments can be sequenced by selecting the templates according to this embodiment. The present-day amplification reactions, such as PCR, are not a limiting factor. One advantage is that no sequence data of the target DNA is needed, nor is any subcloning procedure necessary.

One object of this invention is to provide a kit which comprises a transposon recognizable by a transposase enzyme, the transposase enzyme, a fixed primer hybridizing at a known position in the examined DNA or if the examined DNA is part of a vector for example, adjacent to the examined DNA, and a selective primer including a sequence complementary to the transposon joining end.

One further object of this invention is to provide a kit which comprises a selective primer, a fixed primer, a transposon recognizable by a transposase enzyme, and the transposase enzyme, and wherein the transposon and the transposase enzyme are provided as a transposition complex.

One still further object of this invention is to provide a kit which comprises two different transposon ends recognizable by a transposase enzyme or transposase enzymes, the transposase enzyme or transposase enzymes, and selective primers including a sequence complementary to the transposon joining ends.

These and other objects, together with the advantages thereof over known sequencing methods and kits, which shall become apparent from the specification which follows, are accomplished by the invention as hereinafter described and claimed.

FIG. 1 is a schematic representation of the principle of first (A) and second (B) embodiments of the invention.

FIG. 2 is a schematic representation of the principle of a third embodiment (C) of the invention.

FIG. 3 illustrates the structure of a selective primer, wherein X=additional selective nucleotide.

FIG. 4 shows the result of a selective PCR reaction on an agarose gel as described in Example 1. M=Lambda/HindIII+Phix174/HaeIII DNA marker (Finnzymes Oy, Espoo, Finland). The lane numbers correspond to the reaction numbers in Table 2.

FIG. 5 shows the isolated DNA templates for sequencing on an agarose gel. M=Lambda/HindIII+Phix174/HaeIII DNA marker (Finnzymes Oy, Espoo, Finland). The lane numbers correspond to the fragment numbers marked by arrows in FIG. 4 . Reaction A is performed by both primers, reaction B is performed by the selective primer only.

FIG. 6 shows the results of selective PCR reactions on an agarose gel as disclosed in Example 2. M=Lambda/HindIII+Phix174/HaeIII DNA marker (Finnzymes Oy, Espoo, Finland). “A” refer to the reaction A and “B” refer to reaction B. The numbers corresponds to the DNA fragments selected to the second PCR.

FIG. 7A shows the results of second PCR reactions on an agarose gel as disclosed in Example 2. M=Lambda/HindIII+Phix174/HaeIII DNA marker (Finnzymes Oy, Espoo, Finland). The lane numbers correspond to the fragment numbers marked in FIG. 6 . In FIG. 7B a=reaction with both primers, b=reaction only with the fixed primer, c=reaction only with the selective primer.

FIG. 8 shows the sequence assembly of the Example 2 results.

FIG. 9 shows the results of transposition reaction on an agarose gel as disclosed in Example 3. M=Lambda/HindIII+Phix174/HaeIII DNA marker (Finnzymes Oy, Espoo, Finland). Lane 1=transposition reaction without MuA transposase. Lanes 2 to 6=identical lanes each representing 10 μl samples of transposition reaction. Lane 7 represents linearized pUC19. A=gel before cutting off. B=gel after cutting off.

FIG. 10 shows the results of first PCR in Example 3. M=Lambda/HindIII+Phix174/HaeIII DNA marker (Finnzymes Oy, Espoo, Finland). The lane numbers correspond to the reaction numbers in Table 4.

FIG. 11 shows the results of second selective PCR on a 2% agarose gel as disclosed in Example 3. M=Lambda/HindIII+Phix174/HaeIII DNA marker (Finnzymes Oy, Espoo, Finland). The lane numbers correspond to the reaction numbers in Table 5.

FIG. 12 shows the results of second selective PCR on a 4% Metaphor agarose (FMC) gel as disclosed in Example 3. M=Lambda/HindIII+Phix174/HaeIII DNA marker (Finnzymes Oy, Espoo, Finland). The lane numbers correspond to the reaction numbers in Table 5. The numbers at the side refer to the fragment numbers selected for the third PCR reaction.

FIG. 13 shows the results of third PCR on an agarose gel as disclosed in Example 3. M=Lambda/HindIII+Phix174/HaeIII DNA marker (Finnzymes Oy, Espoo, Finland). The lane numbers refer to the reaction numbers in Table 6 and to the fragment numbers in FIG. 12 .

FIG. 14 shows the sequence assembly of the Example 3 results.

As explained above, this invention provides an in vitro method for selecting templates for DNA sequencing.

The term “DNA sequencing” refers to the determination of the sequence of purine and pyrimidine bases constituting a deoxyribonucleic acid segment. The purines commonly found in DNA are adenine (A) and guanine (G), the pyrimidines commonly found are cytosine (C) and thymine (T). A DNA sequence analysis results in a variation of the four bases designated as ATCGTGC etc.

According to the first embodiment of this invention the method comprises the following steps:

a) A transposition reaction is carried out in the presence of a target DNA, which is a circular DNA or which is part of a circular DNA and in the presence of a transposon.

A “target DNA” is a segment of the DNA of interest to be fully or partially sequenced. The target DNA can range from a few base pairs up to 40 kilo base pairs. The limiting factor for not choosing as target DNA even longer DNA segments is the inability of the present amplification reactions, such as PCR, to produce longer DNA segments. However, in the third embodiment (C) of this invention, the PCR reaction is not a limiting factor and very long DNA segments can be sequenced according to that embodiment.

A “transposon” is a segment of DNA that can insert itself to a target DNA at random or at almost random locations. Transposons used in transposition reactions in vivo normally contain genes coding for enzymes, which are needed in a transposition reaction, but they also contain DNA which is not necessary for the transposition reaction. In reactions in vitro it is sufficient that the transposon used contains only the necessary DNA capable of forming a functional complex with a transposase or integrase enzyme and possibly even with other enzymes needed in a transposition reaction.

It has been shown by Savilahti et al (1995) that a functional transposition complex can be formed using only the ends of phage Mu transposon genome and MuA transposase enzyme.

The term “transposon” is intended to mean a DNA segment or segments which is/are recognizable by a transposase or an integrase enzyme and which is/are capable of forming a functional complex for a transposition reaction.

The term “transposase” is intended to mean an enzyme capable of forming a functional complex with a transposon or transposons needed in a transposition reaction including integrases from retrotransposons and retroviruses.

A “transposition reaction” is a reaction wherein a transposon inserts into a target DNA at random or at almost random sites. Essential components in a transposition reaction are a transposon and a transposase or an integrase enzyme or some other components needed to form a functional transposition complex. The method of this invention is exemplified by employing the transposition complex formed by the ends of Mu genome and MuA transposase enzyme (Savilahti et al., 1995). However, the transposition method is not critical for this invention. All transposition systems capable of inserting amplification primer sites in a random or in an almost random manner are useful in this invention (Craig, 1995). Examples of such systems are Ty1 (Devine and Boeke, 1994, and International Patent Application WO 95/23875), Transposon Tn7 (Craig, 1996), Tn 10 and IS10 (Kleckner et al. 1996), Mariner transposase (Lampe et al., 1996), Tc1 (Vos et al., 1996, 10(6), 755-61), Tn5 (Park .et al., 1992), P element (Kaufmnan and Rio, 1992) and Tn3 (Ichikawa and Ohtsubo, 1990), bacterial insertion sequences (Ohtsubo and Sekine, 1996), retroviruses (Varmus and Brown 1989) and retrotransposon of yeast (Boeke, 1989).

Due to the high sensitivity of the known amplification methods, such as PCR (U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,965,188), the transposition reaction in this embodiment need not be very efficient. Preferably, transposons are inserted into the target DNA in conditions allowing insertion only into a fraction of target molecules to minimize the possibility of having the target DNA with two or more transposon inserts. In case amplification products have sites for selective primer at both ends they are unsuitable for use as sequencing templates.

The transposition reaction is preferably carried out by using a transposition complex comprising only the ends of a transposon DNA and a transposase enzyme resulting in a complete cleavage of the target DNA after the insertion of the transposon.

One transposon insertion linearizes the target DNA, whereas two or more insertions shorten the target DNA giving a smear below the linearized target DNA on an agarose gel. If 5% or less of the target DNA molecules become linearized, the result is sufficient but does not limit the subsequent selective amplification reactions.

b) The target DNA is amplified by using as amplification primers a first primer, the fixed primer, at a known location, and a second primer, the selective primer, at the transposon insertion site.

The “fixed primer” is intended to mean a DNA primer having a sequence complementary to the known sequence in the target DNA or if the target DNA is part of a vector, complementary to the known sequence in the vector adjacent to the insertion site of the target DNA. The vector may be any sequencing vector such as the vectors of the pUC series or their derivatives.

The “selective primer” is intended to mean a primer having a sequence complementary to the joining end of the transposon DNA (complementary to that strand having 5′ end at the joining). The selective primer is in 5′→3′ direction towards the target DNA and has 0 to 10, preferably 0 to 5 additional nucleotides of known identity at its 3′ end (FIG. 3 ).

The term “transposon joining end” means that end of the double stranded transposon DNA, which joins to the target DNA at the insertion site. The transposon joining end does not refer to the strand actually ligating to the target DNA. For example the 3′ end of the transposon MuA joining end ligates to the 5′ end of the target DNA leaving a gap between the 3′ end of the target DNA and 5′ end of transposon joining end.

A selective amplification reaction, such as PCR, is carried out by using the fixed primer and the selective primer as amplification primers. The additional known nucleotides at the 3′ end of the selective primer cause selection of the amplification products. An increasing amount of additional nucleotides improves the selection. In the absence of any additional nucleotides at the 3′ end of the selective primer, the amplification product is a mixture of products of varying sizes making the isolation of products of a specific size very difficult. This is because of the random nature of the transposition reaction in vitro. By adding one additional nucleotide to the 3′ end of the selective primer, the number of possible templates can be decreased to one-fourth compared to a PCR reaction performed without selection. Two additional nucleotides decrease the amount of templates to one-sixteenth, three additional nucleotides to one-sixty-fourth, and so on. A similar approach can be found in Liang P. and Pardee B. (1992) in differential display of mRNA. However, no one has previously suggested the insertion of PCR primer sites to target DNA and the production of sequencing templates for PCR by using selective primers according to the principle disclosed here.

The use of selective primers in the amplification reaction results in specific amplification products varying in size, each size representing one template for a sequencing reaction.

It is preferable to minimize the unspecific PCR products in the selective PCR by performing a “Hot Start PCR”. “Hot Start” can be done by any method known in the art including the addition of DNA polymerase to the reactions after a preheating of samples, isolation of template from DNA polymerase with a wax or using an inactive DNA polymerase that is activated by heat treatment (AmpliTaq Gold, Perking Elmer, USA). Specificity can also be increased by performing an additional PCR before the selective PCR using non selective primer pairs located just before the primers used in selective PCR and transferring small amount of that product into the selective PCR. The method is known as “nested PCR”. Unspecific products can, however, also be used for sequencing if they represent only one DNA template.

To get large PCR products it is preferable to use DNA polymerase mixtures designed for long PCR. Such mixtures, however, often contain 3′-5′ exonuclease activity also called proof reading activity that can cleave the selective nucleotides from the 3′ ends of the selective primers. This problem can also occur if proof reading enzymes like “Vent” or “Deep Vent” from New England Biolabs, USA or Pfu DNA polymerase from Stratagene, USA are used. This phenomenon can be avoided by using phosphorothioate linkages on the last two 3′ bases of the selective primers (De Noronha, C. M. C. and Mullins, J. I., 1992).

c) The amplification products are separated on the basis of their size by methods known in the art such as gel electrophoresis, capillary electrophoresis, HPLC or other well-known methods.

The specific amplification products varying in size are separated on the basis of their size difference by running them on an agarose gel for example, and by picking the separate “bands” into a new amplification reaction, such as PCR, by means of, e.g., a needle or a stick and performing a PCR using the same primers as in the selective amplification. Reaction products of different sizes are obtained, each size corresponding to a separate transposition reaction. The exact position of each PCR product is known in the studied DNA.

By suitably selecting PCR products whose size difference is in the range of the reading capacity of the used sequencing system, normally in the range of 200 to 600 bp, overlapping DNA templates are obtained for sequencing. By using the same fixed primer, it is even possible to sequence a larger DNA segment at one go.

One possible method for selecting suitable templates for sequencing is to use differently labeled fixed and selective primers and to test that both labels are present in the purified amplification product. The labels can be for example radioactive labels or fluorescent labels and the testing can be performed with an appropriate equipment depending on the labels. From the amplification products are selected for use as templates in sequencing those which have both labels.

d) The target DNA is sequenced by using as templates amplification products overlapping each other and varying in size from each other.

The sequencing method can be any sequencing method known in the prior art like the nucleotide specific chemical reaction and cleavage reactions of Maxam and Gilbert (1977) and the primer extension reactions in the presence of nucleotide specific terminators as disclosed by Sanger et al (1977) preferably by using the cycle sequencing method.

According to a second embodiment of this invention the target DNA is a linear DNA or part of a linear DNA. A partial sequence of the target DNA or a DNA adjacent to the insertion site of the target DNA is known enabling the design of a fixed primer having a sequence complementary to the known partial sequence. Accordingly, the position of the fixed primer is known within the target DNA or within the linear DNA comprising the target DNA.

The embodiment B comprises the same method steps as the embodiment A except that there is no linearization step of the target DNA as in the embodiment A.

According to the third embodiment of this invention the method comprises the following steps (FIG. 2 ):

a) Two different sites for amplification primers are inserted to a target DNA by transposition. The transposition reaction is carried out by using transposons that have different ends enabling the design of different selective primers for both ends. The transposon ends can be parts of the same molecule or parts of two separate transposon molecules (Savilahti et al, 1995). In this embodiment it is preferable to have a more efficient transposition reaction than in the embodiments A and B in order to ensure a sufficient number of molecules having two transposon hits.

If required, the other DNA strand can be filled in by DNA polymerase and DNA ligase. Most of the transposition systems leave a nick or gap in one of the strands at their joining site to the target DNA.

b) A selective amplification reaction, such as a PCR reaction, is carried out by using two selective primers as amplification primers. One (first) primer is complementary to one (first) transposon end and the other (second) primer is complementary to the other (second) transposon end. In addition both selective primers have additional nucleotides of known identity at their 3′ end.

The use of selective primers in the amplification reaction results in specific amplification products varying in size, each size representing one template for a sequencing reaction. An increasing amount of additional nucleotides improves the selection.

The selectivity can be accomplished by adding selective nucleotides into the end of only one of the primers. The selectivity can be still further improved by adding selective nucleotides at the end of both primers.

c) The amplification products are separated on the basis of their size by methods known in the art such as gel electrophoresis, capillary electrophoresis, HPLC or other well known methods.

Suitable templates for sequencing can be selected by exposing the amplification products to three parallel amplification reactions. In one of the reactions only one of the selective primers is used, in the other the second selective primer and in the third reaction both selective primers. Only those templates, which are the result of an amplification reaction by both primers are suitable templates for sequencing. However, a control PCR reaction with only one primer sometimes results in a product the same size as a product correctly produced by using both primers. Another method to select suitable templates is to use differently labeled primers and to test that both labels are present in the purified PCR product. The labels can be for example radioactive labels or fluorescent labels and the testing can be performed with an appropriate equipment depending on the labels.

d)The amplification products are situated at random positions at the target DNA. To be able to cover the whole sequence of the target DNA, a sufficient amount of specific amplification products are selected for sequencing templates.

The sequencing method can be any sequencing method known in the prior art like the nucleotide specific chemical reaction and cleavage reactions of Maxam and Gilbert (1977) and the primer extension reactions in the presence of nucleotide specific terminators as disclosed by Sanger et al (1977) preferably using cycle sequencing.

One of the advantages of the third embodiment (C) is that very long DNA segments can be sequenced by selecting the templates according to this embodiment. In this embodiment the present-day amplification reactions, such as PCR, are not a limiting factor, although more sequencing work is needed as compared to the first two embodiments (A and B). Another advantage is that no sequence data of the target DNA is needed nor any subcloning procedure is necessary.

One further object of the present invention is to provide a kit for selecting templates for DNA sequencing.

According to one embodiment of the invention the kit comprises:

a transposon comprising a DNA sequence which is recognizable by a transposase enzyme, said transposon DNA and said transposase enzyme being capable of forming a functional complex needed in a transposition reaction,

a transposase enzyme for carrying out the transposition reaction,

a fixed primer having a complementary sequence to the known sequence in the target DNA or if the target DNA is part of a vector, complementary to the known sequence in the vector adjacent to the insertion site of the target DNA, and

a selective primer having a sequence complementary to the transposon joining end and having 1 to 10, preferably 1 to 5 additional nucleotides of known identity at its 3′ end.

According to another embodiment of the invention the kit comprises:

a transposition complex comprising a transposon and a transposase enzyme, said transposon comprising a DNA sequence which is recognizable by said transposase enzyme,

a fixed primer having a complementary sequence to the known sequence in the target DNA or if the target DNA is part of a vector, complementary to the known sequence in the vector adjacent to the insertion site of the target DNA, and

a selective primer having a sequence complementary to the transposon joining end and having 1 to 10, preferably 1 to 5 additional nucleotides of known identity at its 3′ end.

According to one further embodiment of the invention the kit comprises:

at least one transposon having two different joining ends comprising a DNA sequence or sequences which is/are recognizable by a transposase enzyme, said transposon and said transposase enzyme being capable of forming a functional complex needed in a transposition reaction;

a transposase enzyme for carrying out a transposition reaction, and

a first selective primer, having a sequence complementary to the first joining end and having 0 to 10, preferably 0 to 5 additional nucleotides of known identity at its 3′ end, and a second selective primer, said second primer having a sequence complementary to the second joining end and having 0 to 10, preferably 0 to 5 additional nucleotides of known identity at its 3′ end, wherein at least one selective primer, said first or said second selective primer has 1 to 10, preferably 1 to 5 additional nucleotides at its 3′ end.

The kit may also comprise other components needed in the transposition reaction. Such components may be other enzymes involved in the transposition reaction, or buffers, salts and/or stabilizers which have been chosen depending on the transposase/integrase system.

According to a preferred embodiment of this invention the transposon comprises the Mu transposon ends, recognizable by the MuA transposase.

The following non-limiting examples illustrate the invention in more detail.

Artificial Sequence

Derived from phage Mu from E. coli

1

tgaagcggcg cacgaaaaac gcgaaagcgt ttcacgataa atgcgaaaac aagcttttcc 60

atctctcctc ccccctg 77

2

21

Artificial Sequence

Primer No. 1. Derived from pUC19 from E. coli

2

agctggcgaa agggggatgt g 21

3

26

Artificial Sequence

Primer No. 2. Derived from pUC19 from E. coli

3

ttatgcttcc ggctcgtatg ttgtgt 26

4

20

Artificial Sequence

Primer No. 3. Derived from phage Mu from

E. coli

4

gtttttcgtg cgccgcttca 20

5

21

Artificial Sequence

Primer No. 4. Derived from phage Mu from

E. coli

5

gtttttcgtg cgccgcttca g 21

6

22

Artificial Sequence

Primer No. 5. Derived from phage Mu from

E. coli

6

gtttttcgtg cgccgcttca ga 22

7

23

Artificial Sequence

Primer No. 6. Derived from phage Mu from

E. coli

7

gtttttcgtg cgccgcttca gag 23

8

24

Artificial Sequence

Primer No. 7. Derived from phage Mu from

E. coli

8

gtttttcgtg cgccgcttca gagt 24

9

25

Artificial Sequence

Primer No. 8. Derived from phage Mu from

E. coli

9

gtttttcgtg cgccgcttca gatct 25

10

20

Artificial Sequence

Primer No. 9. Derived from phage Mu from

E. coli

10

cggcgggagt ttgggaggtt 20

11

18

Artificial Sequence

Primer No. 10. Derived from phage Mu from

E. coli

11

gcggcgggag tttgggag 18

12

29

Artificial Sequence

Primer No. 11. Derived from phage Mu from

E. coli

12

cgctttcgta cttcaagtga atcaataca 29

13

30

Artificial Sequence

Primer No. 12. Derived from phage Mu from

E. coli

13

cgctttcgta cttcaagtga atcaatacag 30

14

31

Artificial Sequence

Primer No. 13. Derived from phage Mu from

E. coli

14

cgctttcgta cttcaagtga atcaatacag a 31

15

32

Artificial Sequence

Primer No. 14. Derived from phage Mu from

E. coli

15

cgctttcgta cttcaagtga atcaatacag ag 32

16

33

Artificial Sequence

Primer No. 15. Derived from phage Mu from

E. coli

16

cgctttcgta cttcaagtga atcaatacag agt 33

17

77

Artificial Sequence

Derived from phage Mu from E. coli

17

tgtattgatt cacttgaagt acgaaagcgt ttcacgaaaa acgcgaaagc agatctaacc 60

tcccaaactc ccgccgc 77