WO2005075678A1 - Determination of genetic variants in a population using dna pools - Google Patents

Abstract

The invention provides a system and methods for detecting genetic variants including resistance or genetic susceptibility to disease in a population of individuals using pooled DNA to identify the presence of the desired variant at a genetic locus or loci. In particular, the present invention provides a method for rapidly and relatively inexpensively eliminating from the screening process groups of individuals that lack the gene variant of interest.

Description

DETERMINATION OF GENETIC VARIANTS IN A POPULATION USING DNA POOLS

FIELD OF THE INVENTION The invention relates to a system and methods for detecting genetic variants, including resistance or genetic susceptibility to disease, in a population of individuals, using DNA pools to identify the presence of the desired variant at a genetic locus or loci. In particular, the present invention relates to a method for eliminating from the screening process groups of individuals that lack the gene variant of interest. BACKGROUND OF THE INVENTION Utilization of genetic markers to provide information on the susceptibility of individuals to diseases or disorders is gaining acceptance as a diagnostic or prognostic tool. The selected marker can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition (e.g. obesity, atherosclerosis, cancer); infection by a pathogenic organism (e.g. virus, bacterium, parasite or fungus); or to provide information relating to identity, heredity, or tissue typing (e.g. MHC major histocompatibility complex antigens), in a plurality of individuals. The analysis of variation among polymorphic DNA provides valuable tools for genetic studies in the development of genetic engineering, medicine, gene mapping and drugs. For example, variations in polymorphic DNA allow one to distinguish one individual of a population from another, or to assess the predisposition of an individual to a heritable disease or trait. Two types of genetic markers widely used in genetic studies are microsatelhtes and single nucleotide polymorphisms (SNPs). Microsatelhtes are genomic regions that are distributed approximately every 30 kilobases throughout the genome and that contain a variable number of tandem repeat sequences of mono, di-, tri-, tetra-, penta-, hexa-, hepta-, octa- or nona-nucleotides. SNPs are found in the human genome approximately every kilobase while in the genome of other species including chicken, plant and yeast, their frequencies are much higher. SNPs and microsatelhtes differ in primary DNA structure, relative genome density and genetic information. For example, SNPs are more suitable than microsatelhtes for genotyping with a high-density of markers because of their distribution and the high sequence specificity possessed by sequences adjacent to the SNP site. On the other hand, microsatelhtes are more informative than SNPs because they are more polymorphic than SNPs; microsatelhtes typically possess a large number of alleles per population compared to only two alleles for SNPs. SNPs can serve as genetic markers for identifying disease genes by linkage studies in families, linkage disequilibrium in isolated populations, association analysis of patients and controls, and loss-of-heterozygosity studies in tumors (Wang et al., Science 280: 1077-1082, 1998). Although some SNPs in single genes are associated with heritable diseases such as cystic fibrosis, sickle cell anemia, colorectal cancer, and retinitis pigmentosa (Kerem et al., Science 245: 1073-1080, 1989; Fearon et al., Cell 61 : 759-767, 1990; Sung et al., Proc Natl Acad Sci USA 88: 6481-6485, 1991), most SNPs are silent inasmuch as they lead to no distinguishable phenotype. They can alter phenotype by either controlling the splicing together of exon from intron-containing genes or changing the way mRNA folds. Recently, there has been increasing knowledge of the genetic basis of SNPs for individual differences in drug response (McCarthy et al, Nat Biotechnol 18: 505-508, 2000; Roses, Nature 405: 857-865, 2000). Insights into differences between alleles or mutations present in different individuals can also illuminate the interplay of environment with disease susceptibility. For example, in the p53 tumor suppressor gene, over 400 mutations have been found to be associated with tumors and used to determine individuals with increased cancer risk (Kurian et al., J Pathol 187: 267-271, 1999). All these applications involve the analysis of a large number of samples and will eventually require rapid, inexpensive, and highly automated methods for genotyping analysis. The use of SNPs for human population genetic analyses is disclosed for example in Belouchi et al. US patent application 2002/0155449 and Bader et al US patent application 2003/0101000. Because of the importance of identifying SNPs, a number of gel-based methods have been described for their detection and genotyping. These methods include single strand conformational polymorphism analysis, heteroduplex analysis, denaturing gradient gel electrophoresis, denaturing HPLC and chemical or enzyme mismatch modification assays (Schafer and Hawkins, Nat Biotechnol 16: 33-39,1998; Mashal et al., Nat Genet 9:177-183, 1995; O'Donovan MC. et al., Genomics 52:44-49, 1998). US patent application US2002/0009727 describes various methods for facilitating large- scale SNP identification and new technologies are being developed to replace the conventional gel-based sequencing methods. Perhaps the most widely employed techniques currently used for SNP identification are array hybridization assays, such as allele specific oligonucleotide microarrays in miniaturized assays. There are several other methods emerging for SNP analysis, such as 5' exonuclease assay in which two fluorogenic probes, double-labeled with a fluorescent reporter dye (FAM or TET) and a quencher dye (TAMRA) are included in a typical PCR amplification (Lee et al., Nucleic Acids Res 21 : 3761-3766, 1993). Another popular method that has been successfully applied to the genotyping of SNPs is single nucleotide primer extension (SNuPE), also known as minisequencing. This method can efficiently detect SNPs through the addition of specific nucleotides to a single primer. Several SNP markers can be analyzed in parallel by the use of locus-specific primers and analyzing the allele-specific incorporation of labeled nucleotides (Little, D.P. et al, Eur. J. Clin. Chem. Clin. Biochem 35: 545-548, 1997; Higgins, G.S. et al., BioTechniques 23: 710-714, 1997; Syvanen, A.C. et al., Am J Hum Genet 52:46-59, 1993). Another widely accepted method is called Mass Spectrometry that in general provides a means of "weighing" individual molecules by ionizing the molecules in vacuo and making them volatile.Recently, a combination of single nucleotide primer extension and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS) detection has been developed (Haff et al., Genome Res

7: 378-388, 1997; Griffin et al., Trends Biotechnol 18: 77-84, 2000; Sauer et al., Nucleic Acids Res 28: El 3, 2000). This approach allows the determination of SNP sequences by measuring the mass difference between the known primer mass and the extended primer mass using MALDI-TOFMS. Discrimination of mass differences of less than 1 part in 1 ,000 is required to determine which of the four dideoxynucleotide triphosphate bases (ddNTPs) or deoxynucleotide (dNTPs), reacted to extend the primer. A desired capability of this technique includes the analysis of heterozygotes where two different bases are present at the same nucleotide position. US patent application US2002/0009727 describes MALDI-TOFMS as a mass measurement that requires the discrimination of two mass-resolved species that represent the addition of both bases complementary to those at the SNP site. This requires MALDI-TOFMS methods incorporating high mass resolution capabilities and enhanced sensitivity. Compared to the detection of a fluorescence-labeled nucleotide by non-mass spectrometric methods, mass detection is faster, and less laborious without the need for modified or labeled bases. Mass detection offers advantages in accuracy, specificity, and sensitivity. Recently, a chip-based primer extension combined with mass spectrometry detection for genotyping was performed on a lμl scale in the wells contained within a microchip without using conventional sample tubes and microtiter plates (Tang et al., Proc Natl Acad Sci USA 96: 10016-10020, 1999). This miniaturized method clearly provides another potential for high-throughput and low cost identification of genetic variations. With an ever-increasing database of validated single-nucleotide polymorphisms (SNPs), the limiting factors in genome-wide association analysis have become genotyping capacity and the availability of DNA. Pooled DNA can be efficiently used as a means of efficiently screening SNPs and prioritizing them for further study. This approach reduces the final number of SNPs that undergo full, sample-by-sample genotyping as well as the quantity of DNA used overall (Proc Natl Acad Sci U S A. Dec 24;99(26):16871-16874, 2002). Bader et al disclose the use of pooled DNA in tests of association for quantitative or qualitative traits (U.S. Patent applications 2004/0180376 and 2003/0101000). The inventor previously disclosed the use of DNA pools in preliminary assessment of genetic variation of population and characterizing of a common dominator of a group of individuals (Hillel J. et al., DNA fingerprints in chicken, Proceedings of the 31st British Poultry Breeders' Roundtable Reading pp. 1-11, 1989; Hillel J. et al., Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools Genet Sel Evol. 35(5):533-557, 2003). Pooling of equal amounts of DNA from tens of individuals to thousands of individuals into one sample before genotyping is a valuable means of streamlining large-scale SNP genotyping for disease or rare genetic disorder association studies. The results from pooling are interpreted as a representation of the allele frequency distribution in the individual samples and can be used to validate a candidate SNP as common or rare or merely detect the presence of a particular variation in the pooled DNA sample. Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative disorders associated with misfolding of the prion protein (PrP). Scrapie is a transmissible spongiform encephalopathy (TSE) in sheep and goats that has been reported in countries throughout the world. (Heim D, Kihm U. Rev Sci Tech.;

22(1):179-199, 2003). Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified form of the prion protein (PrPSc). The normal, cellular prion protein (denoted PrPC) is converted into PrPSc through a post- translational process during which it acquires a high beta-sheet content (Prusiner SB

Proc Natl Acad Sci U S A. 10;95(23):13363-13383, 1998). Genetic and molecular properties of PrP isoforms have been explained but the conformational conversion of the PrPC isoform to the PrPSc isoform has not yet been entirely elucidated (Koster T., et al., J Vet Pharmacol Ther. 26(5):315-326, 2003). The level of resistance to Scrapie is determined by combinations of different alleles involving single-nucleotide polymorphisms (SNPs) of the prion gene encoding alternative amino acids at codon positions 136, 154 and 171 (see Table 1). At codon 136, the single SNP causes two amino acid variants; Valine (V) and Alanine (A). At codon 154, the single SNP causes the variants Arginine (R) and Histidine (H). At codon 171 there are two SNPs causing three amino acid variants; Glutamine (Q), Histidine (H) and Arginine (R). Combinations of these amino acid variants produce a total of 5 haplotypes that conventionally are termed alleles of the gene PrP coding for the resistance to Scrapie. U.S. Patent application 2003/0119019 discloses nucleic acid molecules capable of binding a PrP protein isoform thereby distinguishing the isoforms PrPC and PrPSc. The nucleic acid molecules are identified by incubating a pool of nucleic acid molecules with a prion protein isoform and isolating the nucleic acid molecule capable of binding the prion protein isoform. However, the art does not disclose the use of pooled DNA methods for screening for genetic predisposition to a disease associated with PrP misfolding. There is an unmet need for improved methods of genetic analysis of populations, especially methods for analyzing large populations, preferably utilizing single base

DNA variation detection methods. The present invention advantageously eliminates certain genotypes that characterize DNA pools of individuals from a large-scale population, to enable cost effective screening of the remaining individuals.

SUMMARY OF THE INVENTION It is an object of the present invention to provide methods for analyzing the presence or absence of a given genotype in a test population using pooled DNA samples. In particular, the methods of the present invention provide a cost-effective means of screening a population of individuals in order to locate the carriers of a mutation or allele of interest. According to various embodiments the methods of the invention can be used for screening a population of humans, non-human mammals, birds or plants. According to a particular embodiment the methods of the invention can be used for screening a population of domestic animals. It is a further object of this invention to develop a test that utilizes genetic information to predict disease susceptibility and to eliminate undesirable individuals from a large-scale population of animals. The instant invention utilizes processes for detecting a particular nucleic acid sequence in a biological sample. Depending on the sequence to be detected, the processes can be used, for example, to diagnose (e.g. prenatally or postnatally) a genetic disease or chromosomal abnormality; a predisposition to a disease or condition (e.g. TSE, obesity, atherosclerosis, cancer); infection by a pathogenic organism (e.g. virus, bacterium, parasite or fungus); or to provide information relating to identity, heredity, or histocompatibility (e.g. MHC typing), in a plurality of individuals. According to one aspect the invention is suitable for screening of either related or unrelated individuals. These methods are exemplified herein using the principles disclosed for the genotyping of domestic farm animals for Scrapie. These methods are applicable for any other animal population at risk for this or other diseases. The present invention provides a method of screening a test population for individuals having a genotype of interest comprising the steps of: a) generating pooled DNA samples having a substantially equal amount of DNA from each individual within the pool; b) detecting by suitable detection means the presence or absence of a given genotype in each pool; c) eliminating from further screening all individuals represented in any pooled

DNA sample that lacks the gene variant of interest. According to certain preferred embodiments the method further comprises: d) generating de novo DNA pools from remaining individuals. According to certain preferred embodiments the method further comprises: e) repeating steps (a) through (c) on the remaining individuals after each iteration until at least 50%, preferably at least 60% more preferably at least 70% or more of the individuals in the population have been eliminated. According to certain preferred embodiments the method further comprises: f) screening each of the remaining individuals, thereby identifying those individuals that carry the genotype of interest. The present invention relates to a test population comprising a plurality of individuals. According to certain preferred embodiments the method further comprises prior to step (a) assigning an identifying mark or tag to each individual member of a test population. According to certain preferred embodiments the method further comprises prior to step (a) extracting a DNA sample from each individual of the population of interest. According to certain preferred embodiments the method further comprises prior to step (a) determining the concentration of each DNA sample. The methods of the invention may be used with any appropriate detection means for determining the presence of the genotype or mutation of interest. The detection method suitable for use according to the principles of the invention are any detection method that has a sensitivity such that it can detect accurately the presence or absence of the sought genotype or mutation in a pooled sample obtained from at least ten individuals. Preferably the sensitivity will allow detection of one occurrence in 20 individuals or one individual in fifty or even higher sensitivity. Known methods included Mass Spectrometry (MS), pyrosequencing, minisequencing, hybridization and any other method know to one skilled in the art to which the present invention pertains. The present invention will decrease cost and improve the experimental quality needed to achieve genotyping using DNA pooling. Currently preferred methods include mass spectrometric analysis of the pooled

DNA or pyrosequencing of the pooled DNA. As exemplified herein, the method uses the capacity of high sensitivity mass array/spectrometry to efficiently differentiate between given DNA samples and distinguish which of those carry the desirable combination of alleles. For these detection methods the mass of the DNA fragments will suffice to provide means of distinction between genotypes at the selected locus. According to some methods, a combination of specifically designed oligonucleotides for each allele within a marker will allow discrimination between the different genotypes. According to one embodiment preferable detection means of the present invention relates to Mass Array. This system includes a mass spectrometry device which comprises a matrix-assisted laser desorption-ionization-time-of- flight (MALDI- TOF). According to an alternative embodiment of this present invention relates to pyrosequencing. This method is based on the detection of base incorporation by the release of pyrophosphate (PPi). As each nucleotide is added to the growing nucleic acid strand during a polymerase reaction, a pyrophosphate molecule is released. It has been found that pyrophosphate released under these conditions can be detected enzymatically by the generation of light in the luciferase-luciferin reaction. According to the principles of the present invention, it is not required to test each individual of a population or herd. Thus, it is disclosed herein that samples collected from individuals tested may be assigned at random to pooled DNA samples. The number of individuals that may be tested per pool will be determined by the detection means used. The greater the number of individuals that may be pooled the more economical the assay and the greater the benefit derived therefrom. The detection means preferably must be able to detect the tested gene variant using at least ten individuals per pool, more preferably at least 50 individuals per pool, most preferably 100 or more individuals per pool. The second factor that will determine the optimal number of individuals per pool is the frequency of the gene variant or genotype in the population tested. Thus, a frequency in the single percent range will be considered a low frequency, a frequency of one in several hundred or one in a thousand will be considered very low frequency; and a frequency of one in several thousand or less will be considered extremely rare. Using the methods of the present invention it may be calculated that if the incidence or frequency of the genotype of interest is 3-5%, and the sensitivity of the test is capable of detecting at least 1/10 positive individual per pool, then statistically an average of 70% of the pooled samples may be discarded as not encompassing any single individual having the genotype of interest. Thus, it is possible to eliminate 70% of the individual samples, without further testing, saving almost 70% of the total expenditure needed to study each sample individually. The invention also provides methods of using reagents for determining the presence of the specific gene variant. According to one embodiment, the methods of the invention comprise use of nucleic acid sequences complementary to a portion of the sequence of the animal/mammalian PrP gene. In a preferred embodiment, the reagent is specifically hybridizable with the ovine PrP gene (accession number gi:2809230). In another preferred embodiment, the invention is exemplified using nucleic acid sequences as set forth in any one of SEQ ID NOS:l-6. These and other embodiments of the present invention will become apparent in conjunction with the figures, description and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES Figure 1: Calculated number of genotyping reactions required to screen 100 individuals for a very low frequency genotype. Figure 2: Calculated number of genotyping reactions required to screen 100 individuals for a low frequency genotype. Figure 3: Number of genotyping reactions saved (S) as a function of Logio (f) whereas "f ' is the frequency of the gene variant of interest; "R" is the minimal percentage that is detectable by the technology. Figure 4: Number of genotyping reactions saved (S) as a function of Log₁₀ (R). whereas "R" is the minimal percentage that is detectable by the technology; "f ' is the frequency of the gene variant of interest.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods for detecting the individuals in a population bearing a genotype of interest using pooled DNA samples, thereby eliminating the majority of the individuals without resorting to testing each individual in the population. In other words, by testing pooled samples of DNA for the DNA variant of interest, it is now disclosed that all of the individuals in a given pool that is negative for the trait of interest may be eliminated from further screening. Using the methods of the present invention it is economically feasible to test hundreds, thousands or hundreds of thousands of individuals at greatly reduced expenditure in terms of both time and resources than that required in testing each individual in that population. Preferably the expenditure is reduced by at least 50% more preferably 60% or 70%) or more of the cost is eliminated. Definitions and terminology Allele: At a given locus, a particular form of a gene or genotype, specifying one of all the possible forms of the character encoded by this locus. A diploid genome of a heterozygous individual contains two alleles. Genotype: Set of alleles at a specified locus. Polymerase Chain Reaction (PCR) amplification: An enzymatic process resulting in the exponential amplification of specific region of a DNA template. The process uses a thermo-stable polymerase, capable of replicating a DNA template from a primer. In the presence of two primers, the region between them is amplified following this process. Polymorphism: A common variation in the sequence of DNA among individuals. Mass spectrometry: A method by which large molecules, such as DNA fragments, can be identified by precise information on their molecular mass. Techniques for mass analysis known in the art include, but is not limited to, electrospray ionization

(ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry, optionally employing, without limitation, time-of-flight (TOF), quadrupole or Fourier transform detection techniques. Large-scale population: More than 10 individuals, preferably 100 individuals, more preferably 1,000 individuals and most preferably 100,000 individuals. Calculating pool size In this section, a recursive algorithm will be presented for estimating the number of genotyping reactions T needed to screen a population of A individuals for a rare genotype of estimated frequency α. In addition, a procedure will be introduced for deriving the ideal DNA pool size p for any given test iteration. Finally, a computer program for calculating these values as a function of A and α will be presented, and results will be given for the specific case of a population of size 100. Since the ultimate goal is to minimize number of genotyping reactions needed, one may calculate T(A,a) = minT(A,a,p) (1) where T will be minimized through a judicious choice of DNA pool size p. lϊp is the chosen DNA pool size for the first iteration, the total estimated number of genotype reactions necessary to screen the population is equal to the total number of

DNA pools tested in the first testing iteration added to the total estimated number of genotype reactions employed in subsequent testing cycles. One may therefore calculate:

T{A,a,p) = + T'(A,a,p). (2) P wherein the expression denotes the least integer greater than or equal to is the number of DNA pools and hence is equal to the number of genotype

reactions employed in the first testing iteration, while T A,a,p) represents the number of genotype reactions associated for subsequent testing iterations. It is impossible to know a priori for each step exactly how many DNA pools will contain samples from individuals that must be subjected to further testing, and how many DNA pools lack the target genotype. As such, it is only possible to obtain an expected value of T(A,a,p), based upon the weighted average of possible outcomes. Equation (2) therefore becomes the recursive formula

T(A,a,p) = + ___,r, (iP>a r(ip,<x'(a,p)) (3) ι=0 where γ_l is the probability that exactly DNA pools will test positive for the presence of the genotype, the product ip is the number of individuals which warrant further testing given that DNA pools test positive for the genotype, and α' is the estimated genotype frequency among those individuals. Because the parameter γ_l is a probability, it is governed by the identity

while the value of each individual γ_t is given by

The estimated genotype frequency in the set of the individuals subjected to an additional test cycle is derived from the formula _{gt =} l ₊ α(p - l) _{j (6)} Expression (6) reflects the fact that at least one individual in every retained sample pool does indeed display the target genotype. As such, the genotype frequency will necessarily increase after every testing cycle. It is therefore necessary for one who practices the invention to revise the estimate of genotype frequency at the conclusion of every testing cycle. Upon observing how many individuals survive a given testing cycle, and after computing the revised genotype frequency, one who practices the invention would then use the mathematical methods described above to judiciously select the appropriate DNA pool size for the next testing cycle. A computer program based on the algorithms described above has been created to calculate this optimal DNA pool size for any arbitrary population size and genotype frequency. In addition, this program can compute the expected total number of genotyping reactions necessary to screen the entire population. One practicing the invention could therefore use this computer program to select the appropriate DNA pool size for each test cycle. The specific algorithm implemented in this computer program assumes that for each test cycle, the DNA pools are of equal size. This constraint, chosen for the sake of simplicity, limits the possible DNA pool sizes to integers which evenly divide into the population size. Figures 1 and 2 illustrate the expected number of genotyping reactions needed as a function of genotype frequency for a population of size 100. These figures indicate that the invention is most effective for rare genotypes. Thus, we note that for a genotype frequency of 0.005 it is possible, on average, to screen 100 individuals using only 9 genotyping reactions. In the genotype frequency range of 0.03 to 0.05, the average number of tests necessary to screen 100 individuals ranges from 29 to 43 tests. When the genotype frequency reaches approximately 0.3, DNA pooling is of no benefit whatsoever, and it is therefore recommended to test each sample individually. Tables 1 and 2, also created for a population of size 100, display recommended DNA pool sizes for a range of genotype frequencies. In practice, this value would be recalculated using the computer software for each subsequent testing cycle. From these tables, it is clear that the rarer the genotype the larger the recommended DNA pool size, and for a genotype frequency of at least 0.3, it is advised to forgo DNA pooling altogether and to test each sample individually using a pool size of exactly 1.

TABLE 1 : Optimal DNA pool size as a function of genotype frequency for the first testing cycle when screening 100 individuals.

TABLE 2: Optimal DNA pool size as a function of genotype frequency for the first testing cycle when screening 100 individuals

Genotyping genetic variants The present invention relates to a test population comprising a plurality of individuals. The present invention provides methods for identifying genetic markers in individuals of the test population, selected from humans, non-human mammals, non- mammalian animals and plants. According to certain embodiments the population comprises domestic animals. The method comprises (a) providing a given set of polymorphic markers within a population of domestic animals; (b) determining genotypes of at least some individuals in said population for at least some of said polymorphic markers. The terms "genetic marker" and "polymorphic marker" are used interchangeably herein to indicate a known allelic sequence at a known sequence position (or "locus"). It may or may not be part of an expressed gene. Genetic markers may include, but are not limited to: single nucleotide polymorphisms (SNPs), microsatelhtes, mutations, insertions and deletions.

Initial identification of polymorphic marker loci is accomplished by partial sequencing of individual or pooled genomic DNA and a subsequent search for single nucleotide polymorphisms (SNPs). For the purposes of the present invention, SNPs are

DNA sequence variations between individuals that occur when a single nucleotide (A, T, C, or G) in the genome sequence is changed.

In one embodiment of the present invention, the reference population comprises at least 100, preferably 1000, more preferably 10,000, most preferably 100,000 or more individuals. The individuals may be human or non-human mammals, non-mammalian animals (such as avian species) or plants. In another embodiment, the genetic markers are associated with disease susceptibility in breeding programs for domestic animals. Genotypes can be determined by a large number of techniques that allow the detection of the particular genetic marker or gene variant, including for example, methods for detecting SNPs. Some methods for determining genotypes have been reviewed recently (Pui-Yan Kwok, Annu. Rev. Genomics Hum. Genet., 2:235-58, 2001 ; Kirk, B.W., et al., Nucleic Acids Research 30: 3295-3311, 2002; Chee et al., Nucleic Acids Res. 19(12):3301-3305, 1991; Palejwala et al., Biochemistry 32:4105-4111,1993; Michael M. Shi., Clinical Chemistry. 47:164-172, 2001). Such techniques include, but are not limited to, detection on microarrays with fluorescent detection; molecular beacon genotyping; 5' nuclease assays; allele-specific polymerase chain reaction (PCR); allele specific primer extension; arrayed primer extension; homogenous primer extension assays; primer extension with mass spectrometry detection; pyrosequencing; multiplex primer extension; ligation with rolling circle amplification (RCAT); homogenous ligation; multiplex ligation; flap endonuclease assays, for example INVADER™ assays available from Third Wave Technologies (Madison, Wis.) as described in U.S. Pat. No. 6,001,567; mismatch scanning assays, as described for instance in US Patent Application US2003/0129639. Certain methods particularly useful for pooled DNA analysis are described hereinbelow. Genotyping Scrapie resistance in herds of sheep The methods are exemplified in relation to the identification of individuals in a herd of domestic animals that are resistant to the Scrapie prion. According to genetic analyses as are known in the art, the incidence of resistant individuals should fall in the range of 3-5%. In order to identify these 3-5% in a herd of 100,000 individuals the classical methods necessitate 100,000 individual tests. The methods of the present invention should reduce this number by around 66-77%) of the total, respectively. Thus, only 23,000-34,000 individual tests are needed. Prion diseases or transmissible spongiform encephalopathies are a group of closely related transmissible neurodegenerative conditions of humans and animals, all of which are incurable and are associated with misfolding of the prion protein. Conversion of PrPC to aberrant forms such as PrPSc appears to be critical in the transmission and pathogenesis of transmissible spongiform encephalopathies TSEs or prion diseases. PrP genotype controls animal susceptibility and resistance to scrapie and deposition of the disease-associated PrPSc, used as a marker of infection, has the potential to act as a means of identifying TSE-infected animals. Combinations of different alleles involving single-nucleotide polymorphisms

(SNPs) of the prion gene encoding alternative amino acids at codon positions 136, 154 and 171 (Matthews L. et al., Arch Virol. 146:1173-1186, 2001 and Sipos W. et al., Journal of Veterinary Medicine A 49:415-418, 2002), are shown in Table 3. TABLE 3: Five Scrapie alleles, the result of five combinations of amino acid variants at three codons.

A - Alanine, V - Valine, R - Arginine, Q - Glutamine, H - Histidine TABLE 4 - All possible allele combinations of PrP protein with the correlated observed resistance to the disease (Rl- highest resistance to R5 - lowest resistance).

In some cases, information on one of the three codons is sufficient to determine the Scrapie allele, made of the three codons. For instance, when variant V is detected on codon 136, at least one allele is VRQ (see genotypes - form B in Table 5). TABLE 5: Genotypes of Scrapie resistance

Codon Codon Codon GenOiyμes

Genotype # 136 154 171 Risk SNP1 SNP 2 SNP 3 SNP 4 form A form B 1 C G G G AARRRR ARR/ARR Rl C G G C C G G G/C 2 C G A/G G AARRRQ ARR/ARQ R3 c* G* A/G* G/C* 3 c* G* A/G* G/C* AARRRH ARR/ARH R3 C G A/G C 4 C G/A A/G G AARHRQ ARR/AHQ R2 C G/A A/G G/C 5 C/T G A/G G AVRRRQ ARR/VRQ R4 C/T G A/G G/C 6 C G A G AARRQQ ARQ/ARQ R4 7 C G A G/C AARRQH ARQ/ARH R4 8 C G/A A G AARHQQ ARQ/AHQ R3 9 C/T G A G AVRRQQ ARQ/VRQ R5 10 C G A C AARRHH ARH/ARH R4 11 C G/A A G/C AARHHQ ARHAHQ R3 12 C/T G A G/C AVRRHQ ARH/VRQ R5 13 C A A G AAHHQQ AHQ/AHQ R2 14 C/T G/A A G AV HR QQ AHQ/VRQ R4 15 T G A G VVRRQQ VRQ/VRQ R5

* These are the only cases that can be interpreted as either Scrapie genotype 2 or 3.

Although the outcome of a Mass Array analysis, for example, is SNPs information at each of the four sites, it is possible to determine unambiguously each of the 15 genotypes (Table 5), except the very rare case which is indicated in the footnote of Table 5. The present invention will not only increase the throughput of the process at low cost, but this method will also increase the precision and accuracy of genotyping, that is expected to allow cost-effective genotyping of pooled DNA samples.

Figure 3 and 4 describe the number of saved Genotyping reactions (S) due to use of DNA pools, as a function of Log₁₀ (f), or Logio (R). "f ' denotes the frequency of the searched genetic variant. "R" denotes the minimal percentage that is detectable by this method.

The invention also provides the use of reagents for determining the presence of the specific gene variant. According to one embodiment, the reagents include nucleic acid sequences complementary to a portion of the sequence of the animal/mammalian PrP gene. In a preferred embodiment, the reagent is specifically hybridizable with the ovine PrP gene (accession number gi:2809230). The generation of oligonucleotides specifically hybridizable with a nucleic acid template of interest is performed by methods well known in the art, such that a sufficient degree of complementarity or precise pairing allows stable and specific binding between the oligonucleotide and the target nucleic acid molecule under the experimental conditions of the method employed.

Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art, such as enzymatic synthesis or solid-phase synthesis. Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example: Sambrook, J. and Russell, D. W. (2001), "Molecular Cloning: A Laboratory Manual"; Ausubel, R. M. et al., eds. (1994, 1989), "Current Protocols in Molecular Biology", Volumes I-III, John Wiley & Sons, Baltimore, Maryland; Perbal, B. (1988), "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York; and Gait, M. J., ed. (1984), "Oligonucleotide Synthesis"; utilizing solid-phase chemistry, e.g. cyanoethyl phosphoramidite followed by deprotection, desalting, and purification by, for example, an automated trityl-on method or HPLC. The oligonucleotide of the present invention is typically of at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with sequence alterations described hereinabove.

In another preferred embodiment, the invention provides the use of oligonucleotides having nucleic acid sequences as set forth in any one of SEQ ID NOS:l-6 (see Table 7). DNA samples used according to the present invention will be isolated from the above-named sources so as to be essentially undegraded. It will be understood by those with skill in the art that by "essentially undegraded" is meant that the DNA samples will be of sufficient integrity that a mutation or polymoφhism of single copy genes will be detectable by the methods of the invention. Essentially undegraded DNA is isolated by means well known to those with skill in the art as described for instance in US 6,107,026. According to one embodiment exemplified herein below, the present method uses the sensitivity of the Mass Array methodology to detect single nucleotide polymorphisms in a pooled DNA sample composed of substantially equal amounts of

DNA from each individual in the pool, and to distinguish low frequency genotypes out of a large population of related or unrelated individuals in said population.

According to other embodiments the methodology of choice is selected from pyrosequenceing, hybridization, fluorescent DNA sequencing or any other method known in the art. Combination of specifically designed oligonucleotides for an allele marker in the assay will allow determination of the presence or absence of the allele within the DNA sample. In yet another embodiment, the sample is an amplified PCR fragment of the DNA polymoφhism or mutation of interest, including but not limited microsatelhtes, restriction fragment length polymoφhism (RFLP) and amplification fragment length polymoφhism (AFLP). The use of the present invention to perform genotyping with pooled DNA will decrease the expenditure while retaining the informational output of the results of the assay. Therefore, in one embodiment of the present invention, the sample is a mixture of different samples containing a given single nucleotide polymoφhism (SNP) locus from a number of individuals. Another feature of the present invention relates to multiplex analysis by which one has the ability to detect in a sample several SNPs within lOObp, in a single run. Moreover, one skilled in the art can evaluate the allele frequency in pooled DNA, for examples for methods such as Mass Array and pyrosequencing. In carrying out the method of the present invention, genomic DNA can be extracted from whole blood, buccal epithelial cells, saliva stain samples, tissue samples (e.g., mouse tail or a piece from ear), part of an organ, or any other source material as is known in the art. DNA can be extracted from the sample according to means well known in the art (see, e.g., Maniatis, et al., Molecular Cloning; A Laboratory Manual (Cold Spring Harbor Lab, New York, 1982) or by using DNA extraction kit (e.g., Puregene® Gentra Systems Minneapolis, Minn). According to a particular embodiment an alkaline method may be used (Sweet et al., Forensic Sci Int 83: 167-77, 1996; Lin et al., Biotechniques 24: 937-40, 1998; Rudbeck et al., Biotechniques 25: 588-90, 592, 1998). Genomic nucleic acids may be prepared by means well known in the art such as the protocols described in Ausubel, et al., Current Protocols in Molecular Biology, Chapters 2 and 4 (Wiley lnterscience, 1989). As disclosed in US patent No. 6,566,055, in order for mass spectrometry to be a useful tool for screening for polymoφhisms in nucleic acids, several basic requirements need to be met. First, any nucleic acid to be analyzed must be purified to the extent that minimizes salt ions and other molecular contaminants that reduce the intensity and quality of the mass spectrometric signal to a point where either the signal is undetectable or unreliable, or the mass accuracy and/or resolution is below the value necessary to detect the type of polymoφhism expected. Second, the size of the nucleic acids to be analyzed must be within the range of the mass spectrometry-where there is the necessary mass resolution and accuracy. Mass accuracy and resolution do significantly degrade as the mass of the analyte increases; currently this is especially significant above approximately 30,000 Dalton for oligonucleotides (about 100 bases), impacting the detection of single nucleotide polymoφhisms (SNPs) above said mass value. Third, because all molecules within a sample are visualized during mass spectrometric analysis (i.e. it is not possible to selectively label and visualize certain molecules and not others as one can with gel electrophoresis methods) it is necessary to partition nucleic acid samples prior to analysis in order to remove unwanted nucleic acid products from the spectrum. Fourth, the mass spectrometric methods for generalized nucleic acid screening must be efficient and cost effective in order to screen a large number of nucleic acid bases in as few steps as possible. Preferably, the present invention provides mass spectrometric processes for detecting a particular nucleic acid sequence in a biological sample. As used herein, the Mass Array system is an integrated platform that provides an unmatched combination of accuracy, throughput, and simplicity in high-throughput genotyping. The method comprises multiplexed PCR followed by a minisequencing reaction in a single well and detection of the products by MALDI-TOF, as will be described hereinbelow. Genotyping of individual samples, the Mass Array system also supports single reaction allele frequency determination in large sample populations. The method provides processes that can increase the accuracy and reliability of nucleic acid detection by mass spectrometry. In addition, the processes allow for rigorous controls to prevent false negative or positive results. The processes avoid electrophoretic steps, labeling and subsequent detection of a label. In fact, this entire procedure, including nucleic acid isolation, amplification, and mass spectrometry analysis requires only about 2-3 hours time. Moreover, this method allows the nucleic acid fragments to be identified and detected at the same time by their specific molecular weights, an unambiguous physical standard (see for example WO96/29431). The present invention also relates to the Mass Array software that provides realtime genotype analysis, and is the interface to the bioinformatics database. The system is able to automatically align and calibrate the SpectroChip. The SpectroChip is a proven and consistent launching pad for the analysis of DNA samples by MALDI-TOF mass spectrometry. SpectroChips are supplied pre-spotted with a specially formulated MALDI matrix that has been optimized for DNA analysis.

In addition, the Mass Array software reduces individual analysis time for improved data quality and sample throughput. A further advantage of this system is the ability of the MassArray analyzer to simultaneously separate, detect and characterize SNP diagnostic/screening products generated in the PCR extension reaction in less than 1.7 seconds. This rapid analysis complemented by a high-throughput format and up to ten

384 SpectroChips (3840 pads) can be processed automatically in a single run. Another alternative embodiment of this invention is Pyrosequencing that provides a method of identifying a base at a target position in a single-stranded sample DNA sequence. An extension primer, which hybridizes to the sample DNA immediately adjacent to the target position is provided and the sample DNA and extension primer are subjected to a polymerase reaction in the presence of a deoxynucleotide or dideoxynucleotide. The deoxynucleotide or dideoxynucleotide will only become incoφorated and release pyrophosphate (PPi) if it is complementary to the base in the target position. Any release of PPi being detected enzymically, different deoxynucleotides or dideoxynucleotides are being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture and subjected to the polymerase reaction to indicate which deoxynucleotide or dideoxynucleotide is incoφorated. This reaction is characterized in that, the PPi- detection enzyme(s) are included in the polymerase reaction step and in that in place of deoxy- or dideoxy adenosine triphosphate (ATP) a dATP or ddATP analogue is used which is capable of acting as a substrate for a polymerase but incapable of acting as a substrate for a said PPi-detection enzyme. The release of pyrophosphate (PPi) during the polymerase reaction can then be determined by the use of luciferase-luciferin since the amount of light generated is substantially proportional to the amount of pyrophosphate released which in turn, is directly proportional to the amount of base incoφorated. The amount of light can readily be estimated by a suitable light sensitive device such as a luminometer as disclosed in US patent 6,210,891 and reviewed by Shi (Clinical Chemistry 47:164-172, 2001). Pyrosequencing can be used for allele frequency estimation in DNA pools of SNPs with complex sequencing scenarios. Pool size had no significant effect on accuracy and precision (Lavebratt, C et al., Hum Mutat 23(l):92-97, 2004). Another embodiment of the present invention relates to a method of detecting single nucleotide polymoφhisms by Electrospray Ionization spectrometry (ES). The DNA samples generated, for example, by amplifying a region comprising the desired genetic marker or by a primer extension reaction, are dissolved in water or in a volatile buffer, and are injected either continuously or discontinuously into an atmospheric pressure ionization interface (API) and then mass analyzed by a quadrupole (WO9629431). This method/procedure is based on the following stages/phenomena: (a) production of the charged droplets at the ES capillary tip; (b) evolution of the charged droplets due to solvent evaporation and droplet fission caused by Coulombic repulsion of the charges on the droplets; production of the gas phase ion from very small charged droplets by the charge residue model (CRM) or the ion evaporation method (IEM); (c) dependence of the sensitivity in ESMS on the chemical nature of the analyte and its concentration as well as on the concentration of other electrolytes that are present in the solution; qualitative predictions on the sensitivity of the analyte based on the surface activity of the analyte ions; (d) relationship between ions produced in the gas phase and original ions present in the solution; and (e) globular proteins. Another method utilizing Electrospray Ionization spectrometry is described, for example, in U.S. Patent application 2002/0009727. A mixture of target nucleic molecules, the oligonucleotide primers, the nucleic acid polymerizing enzyme, and the nucleotide analogs, each type being present in a first amount, are blended to form an extension solution where the oligonucleotide primer is hybridized to the target nucleic acid molecule to form a primed target nucleic acid molecule and the nucleic acid polymerizing enzyme is positioned to add nucleotide analogs to the primed target nucleic acid molecule at an active site. The oligonucleotide primer in the extension solution is extended by using the nucleic acid polymerizing enzyme to add a nucleotide analog to the oligonucleotide primer at the active site. This forms an extended oligonucleotide primer, wherein the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid molecule at the active site. The amounts of each type of the nucleotide analogs in the extension solution after the extending step are then determined where each type is present in a second amount. The first and second amounts of each type of the nucleotide analog are compared. The type of nucleotide analog where the first and second amounts differ as the nucleotide added to the oligonucleotide primer at the active site is then identified. The steps of extending, determining the amounts of each type of the nucleotide analog, comparing the first and second amounts of the nucleotide analog, and said identifying the type of nucleotide analog added may be repeated, either after repeating the blending with the extended oligonucleotide primer or after determining the amounts of each type of dideoxynucleotide or dideoxynucleotide analog remaining in the extension solution as the new first amount. As a result, the nucleotide at the active site of the target nucleic acid molecule is determined. Also known are apparatus and compositions for carrying out this electrospray method. In addition this system includes an electrospray device which comprises a substrate having an injection surface and an ejection surface opposing the injection surface. The substrate is an integral monolith having an entrance orifice on the injection surface, an exit orifice on the ejection surface, a channel extending between the entrance orifice and the exit orifice, and a recess extending into the ejection surface and surrounding the exit orifice to define a nozzle on the ejection surface. The electrospray system also includes a sample preparation device positioned to transfer fluids to the electrospray device where the sample preparation device comprises a liquid passage and a metal chelating resin positioned to treat fluids passing through the liquid passage. Another advantage of the method is that it permits the use of double-stranded DNA. As a result, there is no need to isolate and separate single-stranded DNA. This method can identify homozygous and heterozygous SNPs in the same experiment (see for example U.S. 2002/0009727). Yet, in another embodiment of the present invention, different method used for the identification of genetic disease is SnuPE also known as minisequencing. This method involves annealing a primer to a template PCR amplicon immediately downstream of, for example, an SNP position. A mix of deoxynucleotide triphosphates (dNTPs) and dideoxynucleotide triphosphates (ddNTPs), or in some cases a mix of ddNTPs alone, are added to the PCR template and primer, along with a DNA polymerase. The polymerase extends the 3' end of the primer by specifically incoφorating nucleotides that are complementary to those contained in the PCR template immediately adjacent to the primer position. Extension terminates at the first position in the template where a nucleotide, complementary to one of the ddNTPs in the mix, occurs. MALDI-TOF-MS- based approaches using minisequencing uses extended primers (shorter than 25 nucleotides) are solid-phase purified and detected by mass. The identity of SNPs is determined by measuring the mass of the extended primer, which is detected at a m/z value specific to the nucleotides added in the extension reaction. In addition to MALDI-TOF-MS- based approaches using minisequencing, such as the Mass Array system described above, other detection methods can be used for minisequencing analyses, including, but not limited to fluorescent and luminous detection methods and other types of mass spectrometry methods well known in the art. In another embodiment of present invention, other types of mutation can be detected by the proposed method. For example, identifying genotypic alterations by the use of restriction fragment length polymoφhism (RFLP) analysis. Using this technique, DNA polymoφhisms can be detected as differences in the length of DNA fragments after digestion with DNA sequence-specific restriction endonucleases. Restriction fragments can then be separated by agarose gel electrophoresis, according to their molecular size, to reveal a pattern of RFLP-related bands. Differences in the length of a particular fragment may result from individual or multiple base substitutions, insertions or deletions. These genotypic changes can be recognized by the altered mobility of restriction fragments on agarose gel electrophoresis. Specific DNA sequences can then be detected by hybridization with a complimentary radioactive probe (see, for example, Botstein et al. Am. J. Hum. Genet. 32: 314-331 (1980) and US Patent No. 6,107,026). The following examples are to be considered merely as illustrative and non- limiting in nature. It will be apparent to one skilled in the art to which the present invention pertains that many modifications, permutations, and variations may be made without departing from the scope of the invention. EXAMPLES Example 1 TABLE 6: Number of genotyping reactions needed to identify rare (3%) genotypes (#6, #20, and #34) in DNA pools of 98 individuals.

According to previous reports known in the art, the frequency of the genotype of interest (ARR/ARR) is 3-5%. To determine the efficiency of the method, given 98 individuals in the test, assume that 3 individuals carry the resistant gene variant and their order numbers are 6, 20 and 34. Thus, seven DNA pools, composed of 14 individuals each, are subjected to DNA pool analysis. In each step some of the pooled DNA samples are discarded and only these that are tested positive to the gene variant are subjected to further detection. It should be mentioned that the remaining of the DNA pools are partitioned in to two equal samples composed of 7 individuals each. In this example, the use of method sensitive to allele copy number allows the exclusion of certain groups of individuals, for example #8-14 and #22-28, from further analysis, as the pooled DNA samples of #1-14, #15-28 and #29-42 are identified as having a single copy of the selected allele in each pool. As shown schematically in Table 6, seven cycles are required in order to detect and verify the 3 samples that carry the resistant genotype, ARR/ARR. A total of 22 genotyping reactions are needed to detect the genotype of interest, without further testing, saving almost 77% of the total expenditure needed to study each sample individually.

Example 2 Collection of blood from each individual will be processed according to Endocrinology, 111; 1149-55, 1982. Ten milliliters (10ml) are extracted from the Jugular vein of each individual and are preserved in a vacutainer tube (Greiner England) that contains anti anti-coagulant K₃EDTA.

Example 3 Extraction of the DNA from mammals - Whole blood from each individual is incubated in red blood cell lysis solution at room temperature for ten minutes, centrifuged for twenty seconds at 13,000xg and the pellet is then resuspended with the red blood cell lysis solution. RNA is discarded by addition of 6μg of RNaseA followed by fifteen minutes incubation at 37°C.

Precipitation of proteins in the sample is achieved by addition of lOOμl of protein precipitation solution. Cell lysate is then centrifuged for three minutes at 13,000xg. The supernatant is transferred to a clean Eppendorf tube containing 100% Isopropanol (300μl) and the DNA sample is then mixed by inversion. The tube is then centrifuged for one minute at 13,000xg, and the DNA pellet is washed with 70% Ethanol followed by air drying and resuspended in 50μl DNA hydration solution. Desirable concentration of the DNA is five to ten μg/μl. Example 4 TABLE 7: List of primer sequences for genotyping Scrapie resistance in herds of sheep.

Example 5 Mass Array Analyses The Mass Array procedure of SNP analyses include the following steps: 1) Extracting of DNA from blood sample of each individual. 2) Dispensing each DNA sample in equal concentration (5ng/well) into the 384 well plate 3) Preparing the PCR reaction mix containing 2.5mM MgCl₂, lx Hotstar Taq buffer, dNTPs mix (200μl/μM of each deoxynucleotide), 0.1 unit of the Hotstar Taq (Qiagen, England), lμM of each of the appropriate forward and reverse primers, and adding to each well 4μl. 4) Transferring the covered plate to the PCR machine for 3 hours. (15min 95°C, 45 cycles: 20sec 95°C, 30sec 56°C, lmin 72°C and followed by 3min 72°C). 5) Adding 2μl of 0.3 units SAP (Shrimp Alkaline Phosphatase mix (Sequenom CA USA). 6) The DNA samples are then incubated 20 min at 37°C followed by 5min 85°C. 7) Removing the plate and centrifuging at 2750g lOsec. 8) Preparing the extension mix: 2.7μM extension primer, specific stop mix (lx buffer with 50μM, of each dNTPs or ddNTPs), 0.58 units Thermo sequenase (Sequenom CA USA). 9) Adding Extension mix to each well, i.e., each DNA sample, spin down the plate at 2750g lOsec, and transferring the plate to the PCR machine for 90 minutes (2min 94°C, 40 cycles: 5sec 94°C, 5sec 52°C, 5sec 72°C. Following this program are 5sec 72°C). 10) Preparing SpectroCLEAN plate (Sequenom CA USA) 11) Adding 80μl water to each well using a robot 12) Subjecting DNA samples to analysis by MALDI-TOF mass spectrometry.

Example 6 TABLE 8: Numerical analysis of saved genotyping reactions.

The percentage of genotyping reactions saved (S) is a function of the percentage of the searched genotype or allele (f), and of the technology resolution (R) - minimal percentage that can be detected by the technology being used. Based on numerical analysis, the following equation has been obtained, within the range off between 10% and 0.01%, and "R" between 20% and 1%. Sj = 100 - 1. OR, - 5.5fj

Number of genotyping reactions saved (S) due to use of DNA pools, as a function of Logio (f), or Logio (R). "f is the frequency of the searched genetic variant, and "R" is the minimal percentage that is detectable by the used technology.

While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, rather the scope, spirit and concept of the invention will be more readily understood by reference to the claims which follow.

Claims

1. A method for screening a population for identifying individuals having a known genetic marker of interest comprising the steps of: (a) generating a plurality of pooled DNA samples having an equal amount of DNA from each individual within each pool; (b) identifying by suitable detection means the presence or absence of the genetic marker in each pool; (c) eliminating from further screening all individuals in a pool that lacks said genetic marker; (d) generating a second plurality of pooled DNA samples from remaining individuals; (e) repeating steps (a) through (c) on the remaining individuals after each iteration, until the number of remaining individuals is less than 50% of the total starting population; (f) genotyping each remaining individual thereby identifying those individuals that carry said genetic marker.

2. The method of claim 1 further comprising prior to step (a) assigning an identifying mark or tag to each individual member of a test population.

3. The method of claim 1 further comprising prior to step (a) extracting DNA from each individual in a test population.

4. The method in claim 1 wherein the detection means reflects the genetic marker frequency in the pooled DNA sample.

5. The method of claim 1 wherein steps (a) through (c) are repeated until the number of remaining individuals is less than 60% of the total starting population.

6. The method of claim 1 wherein steps (a) through (c) are repeated until the number of remaining individuals is less than 70%) of the total starting population.

7. The method of claim 1, wherein the population is selected from: humans, non- human mammals, non-mammalian animals and plants.

8. The method of claim 7 wherein the population is domestic farm animals.

9. The method of claim 1 for screening a related or unrelated population for a genetic marker associated with a condition selected from: a genetic disease; a chromosomal abnormality; a predisposition to a disease or condition; infection by a pathogenic organism; or a genetic marker related to identity, heredity, or histocompatibility.

10. The method of claim 9 for screening a related or unrelated population at risk for a disease.

11. The method of claim 10, wherein the disease is associated with misfolding of the prion protein (PrP) in mammals.

12. The method of claim 11 for screening for Scrapie resistance in domestic animals.

13. The method of claim 12 wherein identifying the presence of said genetic marker in each pool comprises the use of primers having a nucleic acid sequence as set forth in any one of SEQ ID NOS:l-6.

14. The method of claim 1 for confirming the genetic basis of disease predisposition.

15. The method of claim 1 further comprising means for analyzing the results obtained for multiple DNA loci in each DNA pool.

16. The method of claim 1 further comprising the use of an algorithm for determining pool size as a function of genotype frequency and population size in each cycle.

17. The method of claim 1, wherein the presence of said genetic marker is detected using one or more methods selected from the group consisting of: Mass Array, pyrosequencing, minisequencing, Time-Of-Flight mass spectrometry, and Electrospray Ionization mass spectrometry.

18. The method of claim 17 wherein the means is Mass Array.

19. The method of claim 1 further comprising prior to step (a): amplifying at least one target nucleic acid comprising a genetic marker of interest to produce at least one amplified target nucleic acid.

20. The method of claim 12 wherein said detection method comprising means for detection of the presence or absence of the sought genotype or mutation in a pooled sample obtained from at least ten individuals.

21. The method of claim 12 wherein said detection method comprising means for detection of the presence or absence of the sought genotype or mutation in a pooled sample obtained from at least 100 individuals.

22. The method of claim 12 wherein said detection method comprising means for detection of the presence or absence of the sought genotype or mutation in a pooled sample obtained from at least 1000 individuals.

23. The method of claim 14 wherein the means for efficiently differentiating between DNA samples saves at least 60% of total genotyping reactions needed.

24. The method of claim 14 wherein the means for efficiently differentiating between DNA samples saves at least 70% of total genotyping reactions needed.