GB2410796A - Sequence specific DNA analysis - Google Patents

Abstract

A method is provided for sequence-targeted analysis of one or more desired regions of interest within one or more nucleic acid samples comprising the steps of: combining the plurality of nucleic acid samples - if there are more than one - to form a pooled population nucleic acid sample; amplifying the desired region(s) of interest from within the nucleic acid sample by use of the polymerase chain reaction using one or more pairs of primers; optionally normalising the concentration of resultant PCR products; combining them to form a pooled PCR product; creating a nucleotide library from the pooled PCR product; and analysing individual clones isolated from the library. The method may be used for single nucleotide polymorphism (snp) analysis.

Description

24 1 0796 1,1 10] SF,OUENCE-SPECll'IC DNA ANALYSIS hlon I he invention,

relates to a ncthod of a'alysing ONA sequence variation. larlicularly, it relates to netilods using pooled PCR products and Is particularly applicable as a scquecing procedure for screening 1)NA variation hi specific genes or generic regions or at controlled density in the whole genorne. The invention has application in the discovery of SNl's (single nucleotcle polymorphrstns) in organisms including humans and subsequent screening lor those SNI's.

13ackgro rid and Review calf the Art known to the Applicant DNA variation, particularly in tle form ol single nucleolde polymorphisms (SNl's), has lee widely used as an important tnolecular marker in neclical and ag,ricultural applications late current metiloclolog,y for use of Snips comprises three steps. Firstly, Ille dscovcry of SNPs within a population. Secondly, the valitlati.,n ol tile discovered SNIP candidates in relation to their ge''uic ability to identify pheriotypic variations. rilrtily, the devel'pnel of SN1' assays arid the use ol tlcse lo lest samples of interest.

l or the llrst step of SNIP discovery, there arc flour ncillotis: Sh:'lun Seclue'cing (see e.g J. ('raid, Viler, el al 2()()1. Science 291:11()41315), Reduced llepreseltalion Shotgun ) 2 Sequencing (RRS) (see e.g. D Altshuler, et al 20()0 Nature 407: 513-516), Genome l'arltionng (Bela) and sequencing of individual PC1( (pclynerase chain reaction) products. The first three methods comprise hig;II tlroughput sequencing approaches, and mvolve cloning and routine sequencing. 'I'he drawback of these nethcds is that the chr<:mosomal location of the search for SNl's leas a random basis. 'I'he methods cannot locus on any particular group ol' genes or a series of particular chromosone regions. 'I'he fourth metl1od, individual PCIl, can focus on particular regions ol'tile chromosome or genes based on sequences. Ilowever, it loses the higl1 throughput feature for three reasons: (I) the sequencing of individual PCR products has Lo be carried out individually; (ii) the quality ol'sequence data obtained is generally poor; (iii) the PCR reaction has to be optimised Lo produce a single l'CR product I'his is difl'ieult, tirne-eonsuming and failure rates are Leigh. At the present time, however, this method of' sequencing Individual PCR products with a common sequencing primer addition is the Only method available to find new SNl's In a specific region or within specific genes.

For the second step, SNP validation, re-sequencng is the most common method employed. 'I'he procedure is similar to lilat described above for the individual P('R sequencing and is subject to the same constraints and disadvantages.

For the third step, SNIP genotyping or screening, many methods are available in the art, incrusting Ma.ssArray, Pyrosequencing<,'I'ecilman, and Snap-Sllot.

Tile problem remaining is that there is currently no high throughput mottled t'or finding ONA variation in particular genes or regions. Although a few nonillion SNPs have been found in the human genome, and a SNIP haplotype map (I lapMap) has been completed for the human penance, 60'1/u of SNl's discovered in new populations are unknown. thus, using just known SNPs, and assays developed from these, will resmelt in l:ailtre lo discover entirely novel SNl's. 'I'hus, the process of' finding new SNl's in bctl, medical and agricultural sludges continues because these new SNl's are crucial in finding the function of genes.

It is an object ol:'the current Invention to provide a method of discovery and analysis of DNA sequence variation that concubines high tiuouglpuL and gene specilicty fed Sun1mary of the Invention In its broadest aspect, the invention provides a method for sequence-targeted analysis of one or more desired r egions of interest within one or more nucleic acid samples comprising tile steps of: (a) combining the plurality of nucleic acid samples - il'there are more than one - to i'orn to a pooled population nucleic acid sample; (b) anplif'ying the desired region(s) of interest fiom within the one - or the pooled - nueleie acid sample by use ol'tlle polymerase chain reaction (PAR), said PCR reaction using one or more pairs of primers, said pair or pairs ol' primers delinmg said region or regions of interest; (e) optionally normalisng the concentration of 1'(1t products resulting from said ampllieation step; (d) eornbinirl said PCR products to form a pooled PCR product, (e) creating a nueleotide library Tom said pooled P(2R product; (f) analysing individual clones isolated loom said nueleolide library.

Prelerably, the amplification stel1, (b), is carried out using a plurality of primer pairs within the same 1'C'R reaction More preferably, and in any aspect of the invention, the analysis step, (i), comprises the destination of the sequence of DNA, in a plurality ci'cells isolated frown the nucleotde library, derived irons the initial nucleic acid sample I'he nucleic acid samples that f'orrll tle starting point for the method may be ge'omic DNA samples (em taken from animal, bacterial or plant cells) or nay be eDNA samples.

Whilst each individual step of the current hventio is known in itself', the unique conbination ol'steps disclosed herein provides u',expectcd benei'its for DNA analysis, which are discussed below.

General scheme for sequence-targeted analysis l'le following scheme outlines the generalised methodology comprising the current invention. 'I'le scheme relates lo a general situation where it is required to pert'orm a genetic analysis ot'DNA comprising particular regions of interest in a number, m, (where nr-l, 2, 3 etc) ol' populations ol' individuals, each polulalio containing N. individuals (i - 1, 2, .., art) to Key features and benefits ol' tile invention are identified at each step, together with examples lo illustrate the scope of application of Ille invenlcn.

Step 1 Collect T)NA samples prom each inclividtral concerned, within the or each population.

Ex:'mlle A It may be desired to determine the liequency ol' occurrence calf a particular allele within a population ol' plant species and varieties within a particular genus. In this case, there would be one population (i.e. mat) and the "individuals" would comprise each of the distinct species/varieties available.

I',xam/:'le /3 It may be desired to investigate the genetic basis ol' a particular To medical condition of humans. In this case, there Nay be two populations (i e.

m=2) comprishig, say 50 people with the condition, and 65 people without the condition (net N'=50 and N2-65). DNA samples would be obtained from each of these 115 individuals by any number of means that will be apparent to the skilled addressee, and purified to the extent required for selective ampllicalion (e.g. by PAR) Step 9 ('ombine the L)NA samples within each population k' obtain no pooled population DNA samples.

I','xc'n?'le A. For tile case of' example 1.3 in Step I, the 5() samples of' GINA from Jo the first populations would be pooled together to dorm a first pooled population 1)NA sample, and the 65 samples icon tile second population would similarly be pooled together to form a second pooled population DNA sample. / s

Step 3: For each of R regions of interest within the genome, construct a pair of PCR primers, each pair of primers defiling the extremities of tile regions of interest.

1,'xam/1e A In the enlhodinent to be described more fully below (1:'mbodiment 1), 15 regions of interest were identified within the gnome (i.e. R=15), comprising three regions in each of 5 genes. Thus, 15 pairs of primers were constructed. In this example, routine quality control of the following PCR rcaclio,s (see Step 4) revealed that two of the pruner pairs 1cnlecl to give appropriate PCR products, and so those l'CR products were discarded. For the ensuing analysis, therefore, the number of prisoner pairs was reduced accordingly to (i.e. R-13). Small departures fiord the overall protocol, such as this, will be routhlely expected, and easily overcome by the skilled but uninventive addressee.

Methods of constructing primer lairs (i.e. one for each strand of the double stranded DN/) for amplification ol' specific regions of l:)NA will be readily apparent to the skilled addressee. 'l'ypically, a sequence of around 2() nucleotide bases will be used for each primer.

Stop 4: Amplify the regions of' interest in each of the In pooled DNA samples using the R pairs ol'prirners, by either: (a) perfornirlg Fl PCR reactions on the or each pooled ONA samples, each P(.'R reaction using one of the R primer pairs; or (b) combining some o1'lle prhller pairs to lorn1 primer pair groups and perform t:t number ('<R) of l'CR reactions on the or each pooled L)N/L samples, such that each pruner pair is used at least once; OT (c) conbinirlg all the pruner pairs, and perform one 1-C'}{ reaction on the or each pooled l:)NA samples Lam/'le at In the embodiment to be described In more detail below (I'mbodinlent 1), a routine}'CR procedure was enployed, using a proprietary kit (l ligh l fidelity 'I'aq, Norvats). In this case, the initial 15 1'CR reactions were carrred out indepenclenLly.

E.xample B Situations may arise when, say, three primer pairs are to be used (i.c. R 3). Two oi there may define particularly long L)NA sequences, and the third on may define a short sequence. In instances such as this, it may he more convenient (especially t many populations are to he studied) to combine the two primer pairs defining the long sequence and, following the procedure in Step 4(h), perform one 1'C'R reaction using tile combined pruner pairs' and one l'CR reaction ushlg the remaining printer pair. In this way, the PCR reaction conditions (such as melting temperature and annealing time) may be optimism In the first instance for the long sequences and in the second Instance for the short sequence l,xample (' If all the sequences ol interest require similar l'CR reaction conditions, then the procecture in Step 4(c) may be used, combining all the pruner pairs and running just one l'CR reaction.

Ample D If it is known that there is likely to be a higll degree of similarity, or homology, between L)NA sequences in dtl-erent, but perhaps related species, then a single primer may be designed lor use across a slumber of species. For example, a primer may be designed using knowledge of the rice genome, and used to probe other cereals.

so Step 5: If more than cane I'C'R reactions were perlornied on any of tile pooled DNA saml:'lcs, then either: (a) combine the PCIl products resulting from each pooled DNA sample; or (h) assess the relative concentration ot I3C.R product resulting frc,rII each pooled p5 1)NA sample, and combine the lick products hi appropriate ratios so as to produce an approximately equal concentrations of l'(.3R products front each printer pair in each pooled L)NA sample.

Example A: the quantity ol anpllled sequence produced by the l'C'R reaction is Given dependent,n cxperinenLal conditions sued as the quality and quantity of 1)NA polynerase used, tle ati-inity ol the primers for the target sequences, the length ol the target sequence and Ille eoneenlration of nueleotide bases. I bus, in order to optimise the following step of lit rary creation, it is preferable to, ensure that similar amounts of PCRproduced DNA are combined. PCR products may readily be quanll'ied (e.g. by the intensity of bands appearing on an electrophoresis gel), and this may be used to normalise the concentrations. This is the rationale l'or Step 5(b).

Step6: Create a library from the pooled PCR product l'or each of the populations.

Methods for creating "libraries" from mixed populations of DNA liagrnents are well-lulown to those skilled in the art of molecular biology. A number of variants ol' library-creation are known, each of which may have particular to benel'its h1 the application ol'this overall methodology.

Exam/'lc A In the embodiment to be described hi more detail below (.nbodiment 1), Ille DNA fragments were cloned Into a well-known vector (sokl under the Registered 'l'rade Mark "p(:enl" by the Promega C'orpr'ration, USA) and used to transf'ect a culture of Ecoli. 'I'he p('en vector- contains a 1,acZ,rx coding, region allowing selections of recombiriant organism by detection calf the blue/white colour change in the presence of X-(al (5-bromo-4-chloro-3-indolyl p-I)galactopyranoside). rl'he vector also contains '1'7 and SP6 promoters that serve as sequencing prhner binding sites. Other vectors are available (and will doubtless be developed) for construction of DNA libraries, and alternative llankhig sites may be designed to facilitate subsequent processing and analysis of the library.

he library created by Step 6 has a Umber of advantageous features (i) leach positive hitegranl clone in rile library c-,ntahis one copy of the genetic sequence from one ol'the regions ol'nterest.

(ii) Each ol' these sequences may be optionally flanked by a Universal sequencing primer, introduced by suitable choice of vector for the library So creation. This leacis to much improved sequencing qualify, when tle library is subsetinently used Or sequencing studies.

(ui) The library is l-'ree from background DNA that would normally be present in the direct POT product. (A 8

(iv) The library is free from excess primer pair that would normally he present in the direct PC"l' product.

(v)'l'he library is free fiom unpolymerized nucleotide bases that would normally be present in the direct PC'I' product.

(vi) For a given region of interest, the library contains a spectrum ol' genetic sequences in the same ratio as that in the original population. As a result, the library may be used to determine allelic frequencies within its source population.

Also, comparison of allelic lequencies between libraries l'rom different populations provides a measure of the dil'fering allelic frequencies between the to populations themselves Titus, determination of the genetic source of population-e ependant phenotypic trails is facilitated.

(vii) liacl1 gene sequence Cons the region(s) of' interest within the library is present in the same genetic background, and within the same construct. As a result, optional analysis methodologies (e.g. for secluencing) will be substantially the same for each sequence and so such a library provides an ideal starting point for automated analysis.

Step 7 Analyse clones taken from the library.

Lx:mple A In the embodiment to be described in more detail below (t-,mbodiment 1), liron1 each ol' the three libraries constructed, 2()() white colonies (i.e. positive transl'ormants) were selected and sequenced, using the flanking regions as sequencing primers. Methods for sequencing DNA from such libraries are known hi the art. The sequences from each library were assembled using the GAP4 program t'rom the Staden sol'tware suite (MRC Laboratory of Molecular Biology, Cambridge, UK).

The nleth-'d has particular advantages in revealing the association between sequence variation and phenotypic variation. 'I'his is particularly useful in assessment of treatment el'licacy /kictitic)nally, the method has application in the discovery of SNPs, their Jo validation and association with phenotypic traits.

I'ret'elled fmbodilllellts of the Jnvclltioll-lilmbodi1llellt I ( ) 9 This embodiment describes the use of the general methodology for the discovery of DNA sequence variations and allele frequency in a Crohn's L) isease patient population and an ulcerative colitis patient population by comparison with a control population of healthy hldividuals.

ONA samples were collected, with consent, t'ron1 individuals belonging to each of the three populations (i.e. m=3). Inch ol' Ille three populations contained NINA t'rom 2() Individuals (i.e. N,=20, tor i=1,2,3). tulle DN7\ concentration in each sample was ().75 g/litre. The first pooled sampic contained DNA from patients with Crohn's Disease; the to second contained DNA from patients with ulcerative colitis; the third sample contained 1)NA ('Ron heartily volunteers.

Three pairs of primers were designed from the genomic sequence of each of five genes: I IEMI, I,I(BB3, Interleukin 23A, S'l'AT6 and Interferon Gamma. Thus, the methodology used a total of 15 primer pairs (i.e. R-15).

Each pair of primers was designed to amplify 800 to 1200 base pair fragments. A routine l'(:iR procedure (high t-ideLty 'lea, Norvatis) was carried out separately for each pair of primers. Accordingly, 15 PCR reaction products were obtained from each of the three pooled 1)NA samples.

T'hc PCR products from each of these reactions were examined using gel electropiloresis.

I'wo pairs of'primers prepared from the STA'L6 gene 1'ailed to produce PCR products, and so were excluded fTom the study (i.e. R was reduced to 13). Two pairs of primers produced multiple liagnents (two bands and three bands) and were treated as normal PCR proclucts. Inspection of the density ol' bends on the electrophoresis gel allowed the reactions to be divided into three categories, corresponding, to the approximate corcenlralion of the I'CR products 'I'his allowocl normalization of file quantity oi DNA fiagnenis l'rom each reaction. I4'ollowing norrnalisation, the 13 reactions from each pool ol DNA sample were pooled into one tube (the 'pooled 1'CR product') and cloned into a p(,cn (Prome,a ('orporation, USA) vector using routine procedures, and trarisl'ccted into L? coti. This resulted in the construction ol three DNA libraries.

Each of these libraries would therefore contain at least 16 fragments (i. e. 13 reactions, 11 with one band, olIe Title two bands and one with three bands) t'rom its corresponding pooled DNA sample, this sample itself' colItailling all the DNA variation from tile population used to construct it.

It wild be evident that each library could contain more than 16 fragments, as additional fiagnelits witl1 sequence lengths similar to those expected would not leave been readily apparent On the eleciropiloresis gel.

to From each of the three DNA libraries, 200 transformed colonies were selected (using the white/blue reaction supplied by tile pGem vector) and sequenced by routine methodology.

I'Ile sequences loom each library were assembled separately using the GAP4 programme in the Staden software suite (Medical Research C'oulicil L, aboratory of Molecular Biology, ('anhridge, UK).

The DNA sequence variations were revealed loom this GAIN assembling. This analysis revealed 22 variation sites, of which 20 variations were within groups. 45 haplotypes were noted. Six of these haplotypes slowed a skewed frcquelicy distribution between the thiee pcl:'ulations. These are thus ideal candidates for l:'urLher genotyping studies to iclenti (y the genetic association to the two diseases.

Pret'erred l-2mbodililell tS-E'I1 bodimelit 1l as In this example, the sane l)NA samples as described in F'mbodililellL T were used, i c thiee pool DNA samples representing individuals with ('rolill's Disease, ulcerative colitis, and individuals frolic a contiol population.

In Ihi.s embodinelit, 50 pairs of primers wele designed representing 50 locations to cover a region of chiomosolile 12, between micolsatellitc markers D12S368 and D12S1632.

Tlese Makers are at chroniosonie pOSitiOllS 5()947746 and 66834816 In chromosome 12.

Ellis region also contains tle microsatcilite markers L)1251632, D125910 and D12583.

Within these 50 locations or loci, 11 were from the experiment of Embodiment I (the ones giving the single band gel pattern), 26 were frown known genes in addition to those 5 genes used in Embodiment 1, six were front around the microsatellite markers D12S83, D12S85 and D]2S90. A hrtller eight were randomly chosen to 1;11 the gaps. On average, the gap among the 50 selcctecl sequences was 255kb. Tile largest gap was around 88kb and the smallest gap about 50kb.

t.Jsing a similar methodology as that describeci in Embodiment 1, three libraries were constructed alter the PCiR products were checkocl and selected. From the P(:R products, to 48 I'CR reactions were selected for use in library creation, and 57 DNA fragments were expected to be represented.

From each of these three libraries, 6()0 transiormant colonies were selected and sequencecl. the sequences were analysed in the same way as in Embodiment 1. In total, 231 haplotypes were noted, 20 ol these showing a skewed liequency distribution between tle three population groups, including the 6 haplotypes identified in Embodiment 1. ]2

Claims (2)

1. method for sequence-targeted analysis of one or more desired regions of Interest within one or more nucleic acid sarilp]es comprising the steps of' (a) conbining the plurality of nucleic aeicl samples - if there are more shall one -- to form a pooled population nucleic acid sample; (b) amplifying the desired region(s) of interest from within the one - or the pooled -- nucleie acid sample by rise ol' the polymerase chain reaction (I'CR), said PCR reaction lo using one ol more pairs of primers, said pair or pairs of primers defining said region or regions of interest, (e) optionally norinalising the concentration of l'CR products resulting thrum said amplificati(-,rl step; (cl) colilbblilig said P(:R products to l'orm a pooled l'CR product; (e) creating a nucleotide library tiom said pooled P('R prctiuet; (l) arialysing individual clones isolated fiom said ntieleotrde library.

2. The methcti of Claim I wherein the amplification step, (b), is carried out using a plurality of primer pairs within the same PC11 reaction.

3 'I'lle method ol' any of any preceding claim wherein the analysis step, (f), comprises the deterilliliatioll ol'the sequeliee ol'DNA, in a plurality ol'eells isolated from the nueleotide library, derived frolic tile initial nucleic acid sa.nple.