SUMMARY OF THE INVENTION |
The present invention relates to a metho sequence utilising interlaced nesting pr sequencing purposes and, in particular, when applied to sequencing has been coin Interlaced Nesting (ASIN). |
d of amplification of a nucleotide
imers. The method may be used for
for genome sequencing. The method
ed Amplification and Sequencing by
|
According to a first aspect, the present ying a nucleotide sequence of interest w at least one region of known sequence, w comprises, 3' to 5': a first known regio the first and second known regions are i wherein the method comprises: |
invention provides a method of amplif- herein the nucleotide sequence comprises herein the region of known sequence n and a second known region wherein mmediately adjacent each other and |
1) a first amplification step comprising |
at least |
(a) as a template, a sequence comprising of interest; and |
at least the nucleotide sequence |
(b) one first primer having, 5' to 3': a corresponding to the first known region; second known region of the nucleotide of |
5' tag sequence; a degenerate sequence and a sequence complementary to the interest; |
which amplification step generates a fir |
st amplification product; and |
2) a second amplification step comprisin |
g at least |
(a) as a template, the first amplificati |
on product; and |
(b) one second primer having, 5' to 3': complementary strand of the 5' tag seque complementary to the first known region which amplification step generates a sec |
a sequence which hybridises to the nce of the first primer and a sequence of the nucleotide sequence of interest; ond amplification product. |
Preferably, the amplification step is a |
polymerase chain reaction (PCR). |
Preferably, in the first and second prim and the sequence which hybridises to the sequence of the first primer, are the sa |
ers respectively, the 5' tag sequence complementary strand of the 5' tag me. |
Preferably, the 5' tag sequence is a vec the 5' tag sequence is derived from M13 CRIPT, or pBELOBACII. However, the skill that any appropriate 5' tag sequence can |
tor specific sequence. More preferably, phage, pUC18, pBR322, pGEM.RTM., BLUES- ed addressee will, of course, understand be used. |
Preferably, the 5' tag sequence is 10 to the 5' tag sequence is 15 to 18 bases in sequence is 15 bases in length. |
25 bases in length. More preferably, length. Most preferably, the 5' tag |
Preferably, in the first primer, the seq known region is 4 to 8 bases in length. the sequence complementary to the second Preferably, in the first primer, the deg sequence complementary to the second kno is 6 to 12 bases in length. Most prefera sequence together with the sequence comp of the nucleotide of interest is 9 bases |
uence complementary to the second More preferably, in the first primer, known region is 6 bases in length. enerate sequence together with the wn region of the nucleotide of interest bly, in the first primer, the degenerate lementary to the second known region in length. |
Preferably, in the second primer, the se known region of the nucleotide sequence More preferably, in the second primer, t known region of the nucleotide sequence Preferably, the 5' tag sequence, or a se entary sequence of the 5' tag sequence, atively, the second primer is used as a |
quence complementary to the first of interest is 1 to 5 bases in length. he sequence complementary to the first of interest is 3 bases in length. quence which hybridises to the complem- is used as a sequencing primer. Altern- sequencing primer. |
In one embodiment, the first and second primers and two second primers respectiv acts as a reverse primer in the first am primers acts as a reverse primer in the be clear to the skilled addressee that t first primers may be the same or differe |
amplifications comprise two first ely, wherein one of the first primers plification step and one of the second second amplification step. It will he 5' tag sequence on each of the nt. |
It will also be clear to the skilled add performed sequentially or simultaneously |
ressee that steps 1) and 2) may be . |
According to a third aspect, the present obtained by a method according to the fi |
invention provides a product when rst or second aspect. |
According to a fourth aspect, the presen 5' to 3': a 5' tag sequence: a degenerat ce, wherein the sequences are immediatel the predetermined sequence is 4 to 8 bas is 6 bases in length. Preferably, the de predetermined sequence is 6 to 12 bases is 9 bases in length. |
t invention provides a primer having, e sequence: and a predetermined sequen- y adjacent to each other. Preferably, es in length and more preferably it generate sequence together with the in length and more preferably, it |
According to a fifth aspect, the present 5' to 3': a sequence which hybridises to tag sequence of a primer according to th sequence. Preferably, the predetermined and more preferably it is 3 bases in len |
invention provides a primer having, the complementary strand of the 5' e fourth aspect, and a predetermined sequence is 1 to 5 bases in length gth. |
The 5' tag sequence of the primers of th a vector specific sequence such as that pGEM, BLUESCRIPT, or pBELOBACII. Prefera fifth aspects are 10 to 25 bases in leng in length and most preferably 15 bases i |
e fourth and fifth aspects may be derived from M13 phage, pUC18, pBR322, bly the primers of the fourth and th, more preferably 15 to 18 bases n length. |
According to a sixth aspect, the present ising at least two primers wherein the p ding to the fourth or fifth aspect. The which at least one primer is a primer ac least one primer is a primer according t |
invention provides a primer set compr- rimers are selected from primers accor- primer set may be a primer set in cording to the fourth aspect and at o the fifth aspect. |
According to a seventh aspect, the prese comprising primers according to the four |
nt invention provides a primer library th aspect. |
According to an eighth aspect, the prese comprising primers according to the fift |
nt invention provides a primer library h aspect. |
According to a ninth aspect, the present comprising primers according to the four |
invention provides a primer library th or fifth aspect. |
According to a tenth aspect, the present comprising primer sets according to the |
invention provides a primer library sixth aspect. |
The libraries may be produced by any mea art. Preferably, the libraries are produ phosphoramidite method. |
ns known to persons skilled in the ced by chemical synthesis using the |
According to an eleventh aspect, the pre a primer according to the fourth or fift |
sent invention provides a kit comprising h aspect. |
According to a twelfth aspect, the prese a primer set according to the sixth aspe |
nt invention provides a kit comprising ct. |
According to a thirteenth aspect, the pr ing a primer library according to any on Preferably, the kit is used in the metho will be appreciated that the kit may fur enzymes, and a sample of one or more sui |
esent invention provides a kit compris- e of the seventh to tenth aspects. d of the first or second aspect. It ther include optional buffers, diluents, table reverse primers. |
According to a fourteenth aspect, the pr of amplifying and sequencing a nucleotid nucleotide sequence comprises at least o the region of known sequence comprises, 3 bases and a second known region of 6 b known regions are immediately adjacent e ises: |
esent invention provides a method e sequence of interest wherein the ne region of known sequence, wherein 3' to 5': a first known region of ases wherein the first and second ach other and wherein the method compr- |
1) a first amplification step comprising |
at least |
(a) as a template, a sequence comprising of interest; and |
at least the nucleotide sequence |
(b) one first primer having, 5' to 3': a corresponding to the first known region; second known region of the nucleotide of |
5' tag sequence; a degenerate sequence and a sequence complementary to the interest; |
which amplification step generates a fir |
st amplification product; and |
2) a second amplification step comprisin |
g at least |
(a) as a template, the first amplificati |
on product; and |
(b) one second primer having, 5' to 3': primer and a sequence complementary to t sequence of interest |
the same 5' tag sequence as the first he first known region of the nucleotide |
which amplification step generates a sec |
ond amplification product; |
3) sequencing the second amplification p a sequencing primer. |
roduct using the 5' tag sequence as |
According to a fifteenth aspect, the pre amplifying and sequencing a nucleotide s tide sequence comprises at least one reg region of known sequence comprises, 3' t known region wherein the first and secon each other and wherein the method compri |
sent invention provides a method of equence of interest wherein the nucleo- ion of known sequence, wherein the o 5': a first known region and a second d known regions are immediately adjacent ses: |
1) an amplification step comprising at l |
east |
(a) as a template, a sequence comprising of interest; and |
at least the nucleotide sequence |
(b) one primer having, 5' to 3': a 5' ta corresponding to the first known region; second known region of the nucleotide of |
g sequence; a degenerate sequence and a sequence complementary to the interest; |
which amplification step generates an am |
plification product; and |
2) a sequencing step in which the amplif a sequencing primer. |
ication product is sequenced using |
Preferably, the sequencing step of the f (a) as a template, the amplification pro |
ifteenth aspect comprises at least duct; and |
(b) the sequencing primer having, 5' to the complementary strand of the 5' tag s the amplification step, and a sequence c of the nucleotide of interest. |
3': a sequence which hybridises to equence of the primer utilised in omplementary to the first known region |
According to a sixteenth aspect, the pre amplifying and sequencing a nucleotide s tide sequence comprises at least one reg region of known sequence comprises, 5' t known region wherein the first and secon each other and wherein the method compri |
sent invention provides a method of equence of interest wherein the nucleo- ion of known sequence, wherein the o 3': a first known region and a second d known regions are immediately adjacent ses: |
1) an amplification step comprising at l |
east |
(a) as a template, a sequence comprising of interest; and |
at least the nucleotide sequence |
(b) one primer having, 5' to 3': a 5' ta corresponding to the first known region: second known region of the nucleotide of |
g sequence: a degenerate sequence and a sequence complementary to the interest; |
which amplification step generates an am |
plification product; |
2) a sequencing step in which the amplif a sequencing primer. |
ication product is sequenced using |
Preferably, the sequencing step of the s (a) as a template, the amplification pro |
ixteenth aspect comprises at least duct; and |
(b) the sequencing primer having, 5' to the complementary strand of the 5' tag s the amplification step, and a sequence c of the nucleotide of interest. |
3': a sequence which hybridises to equence of the primer utilised in omplementary to the first known region |
Preferably, the amplification step of th chain reaction. In the primer utilised i sequencing primers respectively, the 5' hybridises to the complementary strand o utilised in the amplification step, may sequence may be a vector specific sequen pGEM, BLUESCRIPT, or pBELOBACII. Prefera 25 bases in length, more preferably 15 t 15 bases in length. In the method of the sed in the amplification step, the seque region may be 4 to 8 bases in length and In the primer utilised in the amplificat together with the sequence complementary nucleotide of interest is 6 to 12 bases in length. In the sequencing primer, the known region of the nucleotide sequence length, more preferably 3 bases in lengt |
e sixteenth aspect is a polymerase n the amplification step and in the tag sequence and the sequence which f the 5' tag sequence of the primer be the same or different. The 5' tag, ce such as M13 phage, pUC18, pBR322, bly, the 5' tag sequence is 10 to o 18 bases in length and most preferably sixteenth aspect, in the primer utili- nce complementary to the second known more preferably, 6 bases in length. ion step, the degenerate sequence to the second known region of the in length, more preferably 9 bases sequence complementary to the first of interest may be 1 to 5 bases in h. |
It will be clear to the skilled addresse tion can be used as forward and reverse can be used to amplify (and sequence) an is also clear that since large parts of and the entire genome sequence of others used to generate a library or an array o frames of an organism. |
e that, since the primers of the inven- primers, the methods described above y desired nucleotide sequence. It the genome sequence of some organisms, is known, the invention could be f sequences of, say, open reading |
According to a seventeenth aspect, the p produced by a method according to any on aspects. |
resent invention provides a product e the fourteenth to the seventeenth |
According to an eighteenth aspect, the p sing a product of the third or eighteent |
resent invention provides a kit compri- h aspects. |
According to a nineteenth aspect, the pr according to any one the first or second nth aspects when used in a method of pri |
esent invention provides a method aspects or the fourteenth to seventee- mer walking. |
According to a twentieth aspect, the pre according to the third aspect, when used |
sent invention provides a product in a method of primer walking. |
According to a twenty-first aspect, the according to the fourth or fifth aspect, According to a twenty-second aspect, the set according to the sixth aspect, when According to a twenty-third aspect, the library according to any one of the seve a method of primer walking. |
present invention provides a primer when used in a method of primer walking. present invention provides a primer used in a method of primer walking. present invention provides a primer nth to tenth aspects, when used in |
According to a twenty-fourth aspect, the according to any one of the eleventh to method of primer walking. |
present invention provides a kit thirteenth aspects, when used in a |
General laboratory procedures not specif can be found in the general molecular bi Sambrook et al. (1989) Molecular Cloning Harbor Laboratory:Cold Spring Harbor, N. |
ically described in this specification ology texts including, for example, : A laboratory Manual. Cold Spring Y. |
In the context of the present specificat on" and its acronym "PCR" are used accor understood by those skilled in the art. in common molecular biology textbooks an For example PCR Technology: Principles a (1989) Ed. H. A. Erlich Stockton Press, |
ion the terms "polymerase chain reacti- ding to their ordinary meaning as Examples of PCR methods can be found d reference manuals used in the art. nd Applications for DNA Amplification New York. |
In order to optimise the PCR amplificati concentrations and ratios. Selection of appreciated and obtainable by persons sk |
on, the primers can be used at different these and other variables would be illed in the art. |
In the context of the present invention, tion" should be construed in the sense o and "the production of at least one copy |
the terms "to amplify" and "amplifica- f "to produce at least one copy of" of". |
In the context of the present invention, be construed in the sense of any primer In the context of the present invention, acid", "nucleotide sequence" and "templa to their ordinary meaning as understood |
the term "sequencing primer" should which can initiate a sequencing reaction. the terms "oligonucleotide", "nucleic te" should be construed according by the skilled addressee. |
In the context of the present specificat be construed according to its ordinary m in the art, i.e. in the sense of utilisi obtain nucleotide sequence from an unkno nucleotide sequence thus obtained to obt a further flanking region. The process c Unless the context clearly requires othe and the claims, the words "comprise", "c construed in an inclusive sense as oppos that is to say, in the sense of "includi |
ion, the term "primer walking" should eaning as understood by those skilled ng a known nucleotide sequence to wn flanking region and utilising the ain further nucleotide sequence from an be repeated any number of times. rwise, throughout the description omprising", and the like are to be ed to an exclusive or exhaustive sense: ng, but not limited to". |
BRIEF DESCRIPTION OF THE DRAWINGS |
FIG. 1 is a schematic outline of an ASIN invention. The spotted region represents region represents the first known region second known region. |
procedure according to the present the 5' tag sequence, the striped , and the grey region represents the |
FIG. 2 shows a diagrammatic example of t products by a nesting reaction of the AS invention. The first and second known re interest are indicated. |
he suppression of non-desired PCR IN procedure according to the present gions of the nucleotide sequence of |
FIG. 3. The 1727 bp E. coli fruR region and EK10FS. The annealing sites of EK10R 8 nucleotide long annealing sites of the boxed. PCR amplification using the ASIN will result in a PCR products of 1108 bp |
PCR amplified with the primers EK10R and EK10FS are underlined. The three first round ASIN primer TTCCTG are second round primer CTAG-GC and CTAG-TA and 595 bp, respectively. |
FIG. 4. ASIN PCR reactions using the fir round primers CTAG-GC and CTAG-TA. Lane 1078, 872 and 603 bp). Lane 2: 6 .mu.l o the second round primers CTAG-TA and EK1 product. Lane 4: 6 .mu.l of the ASIN PCR primers CTAG-GC. |
st round primer TTCCTG and the second 1: 150 ng of f174 DNA marker (1353, f the ASIN PCR amplification using 0R. Lane 3: 1 .mu.l of the fruR PCR amplification using the second round |
FIG. 5. The 3172 bp E. coli fusA region. sites of the first round ASIN primers 4G ation using the ASIN second round primer result in PCR products of 509 bp and 144 |
The three 9 nucleotide long annealing 1, 4G3 and 4G6 are boxed. PCR amplific- 4G9 and 4G12 or 4G7 and 4G12 will 0 bp, respectively. |
FIG. 6. fusA ASIN PCR and control reacti DNA marker (8557, 7427, 6106, 4899, 3639 992, 710, 492, 359.81 bp) Lane 2: 7 .mu. tion using primers 4G3 and 4G6. Lane 3: using primers 4G9 and 4G12. Lane 4: 7 .m cation using primers 4G 1 and 4G6. Lane using primers 4G7 and 4G12. |
ons. Lane 1: 150 ng of Sppl/EcoRI , 2799, 1953, 1882, 1515, 1412, 1164, l of the control ASIN PCR #1 amplifica- 3 .mu.l of the ASIN PCR #2 product u.l of the control ASIN PCR #1 amplifi- 5: 1 .mu.l of the ASIN PCR #2 product |