WO2003050294A2

WO2003050294A2 - Genotype analysis using human hair shaft

Info

Publication number: WO2003050294A2
Application number: PCT/US2002/039228
Authority: WO
Inventors: Ie-Ming Shih
Original assignee: The Johns Hopkins University
Priority date: 2001-12-06
Filing date: 2002-12-06
Publication date: 2003-06-19
Also published as: WO2003050294A3; AU2002360521A1

Abstract

The present invention provides novel methods for performing genetic analysis using DNA isolated from human hair shafts.

Description

GENOTYPE ANALYSIS USING HUMAN HAIR SHAFT

Related Applications

This application claims the benefit of U.S. Provisional Application Serial No. 60/337,316, filed December 6, 2001, the entire contents of which are incorporated herein by this reference.

Background of the Invention

It is widely believed that the identification of genes causing or predisposing to a host of human diseases will revolutionize the practice of medicine and public health in post- genomic era. Study of the association between genotype and disease risk will also help in the understanding of the genetic and molecular basis of disease. Although lymphocytes and buccal swabs have been routinely used as the sources of genomic DNA for genotype analysis (Zheng, S. et al. (2001) Cancer Epidemiol. Biomarkers Prev. 10:697-700; Garcia-Closas, M. et al. (2001) Cancer Epidemiol. Biomarkers Prev. 10:687-96), a highly compliant, totally non-invasive and convenient system for genotyping is of great interest, especially for population-based genetic screening. Human hair has long been known to contain genomic DNA (Higuchi, R. et al. (1988) Nature 332:543-6) and represents one of the potential DNA sources for non-invasive DNA sampling. Several studies have examined such possibility using dot-blot hybridization (Higuchi, R. et al. (1988) Nature 332:543-6; Uchihi, R. et al. (1992) J. Forensic Sci. 37:853-9), microsatellite markers (Sasaki, M. et al. (1997) Forensic Sci. Int. 90:65-75), and PCR - restriction fragment length polymorphism (PCR-RFLP) (Tanigawara. Y. et al. (2001) Ther. DrugMonit. 23:341-6). Based on these studies, the analysis of hair genomic DNA is fraught with technical difficulties. Such hair DNA, especially obtained from the terminals, is very limited is quantity, and accordingly, the genotyping results may not be reproducible, thus limiting its wide clinical application. Because of the limitations and variability of using hair genomic DNA, accurate analysis currently requires enrichment of DNA purified from the root portion, because the root region and hair follicles contain more abundant genomic DNA needed for multiple assays (Higuchi, R. et al. (1988) Nature 332:543-6; Uchihi, R. et al. (1992) J. Forensic Sci. 37:853-9; Sasaki, M. et al. (1997) Forensic Sci. Int. 90:65-75). However, plucking hair at the root may not be desirable for many individuals for a number of reasons (e.g., because it may be painful), and in many cases, the root may not be available at all (for example, hair found at crime scenes). Therefore, there is a major need in the art for a reliable, reproducible method for isolating genomic DNA from the hair shaft in sufficient quantities to perform genetic analysis.

Summary of the Invention

The present invention is based, at least in part, on the discovery of a method for performing genetic analysis using the DNA isolated from hair shafts, rather than hair roots. The methods of the invention provide a reliable, reproducible method for isolating genomic DNA from the hair shaft in sufficient quantities to perform genetic analysis.

Accordingly, the invention provides methods of determining the genotype of a subject at at least one predetermined locus comprising isolating at least about 40 pg of genomic DNA from the hair shaft(s) (e.g., the cephalic, pubic, and/or body hair shafts) from the subject, and testing said genomic DNA to determine the genotype of the subject at the predetermined locus. Preferably, the methods of the invention do not require the presence of the hair root. In a further preferred embodiment, the subject is a human. In other embodiments, the subject may be any mammal or other animal that has hair, e.g., a cat, dog, cow, horse, pig, goat, monkey, chimpanzee, mouse, rat, or rabbit.

In another embodiment, the predetermined locus is amplified (e.g., by PCR) prior to testing. In another embodiment, the predetermined locus is a single-nucleotide polymorphism (e.g., the APO-E 112 polymorphic site, the APO-E 158 polymorphic site, or the GNB3 (C825T) polymorphic site). In further embodiments, the methods of the invention further comprise determining the genotype of the subject at at least a second and/or third predetermined locus. hi another embodiment, the methods of the invention comprise the use of at least two molecular beacons capable of differentiating between the different alleles of the predetermined locus. In other embodiments, the methods of the invention comprise direct sequencing of the predetermined locus, RFLP (restriction fragment length polymorphism) analysis, SCAR (sequence characterized amplified region) analysis, SSLP (simple sequence length polymorphism) analysis, SSR (simple sequence length repeat) analysis, STS

(sequence-tagged site) analysis, microsatellite marker analysis, STSM (sequence-tagged microsatellite marker) analysis, minisatellite marker analysis, polymorphic repeat analysis, RAPD (random amplified polymorphic DNA) analysis, AFLP (amplified fragment length polymoφhism) analysis, SAMPL (selective amplification of microsatellite polymoφhic loci) analysis, and/or CAPS (cleaved amplified polymoφhic sequence) analysis.

In a preferred embodiment, the DNA is isolated from the hair shaft(s) using a method comprising treating the hair shaft with proteinase K. In a further preferred embodiment, the DNA is isolated from the hair shaft(s) using a method comprising selectively binding the DNA to a silica-gel membrane that does not substantially bind non-DNA contaminants. In another preferred embodiment, the DNA is isolated from about 20 mg of hair.

In another preferred embodiment, the invention provides kits for determining the genotype of a subject at at least one predetermined locus (e.g., an SNP such as the APO-E 112 polymoφhic site, the APO-E 158 polymoφhic site, or the GNB3 (C825T) polymoφhic site) comprising reagents suitable for isolating at least about 40 pg of genomic DNA from at least a portion of a hair shaft from the subject, at least two nucleic acid primers which specifically amplify the predetermined locus, and at least two molecular beacon molecules which specifically differentiate between two different alleles of the predetermined locus. In further preferred embodiments, the kits of the invention include primers and molecular beacons specific for at least a second or third predetermined locus.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings

Figure 1 depicts the amount of DNA isolated from the hair shafts from different individuals. The amount of hair shaft DNA ranges from 0.02 to 20 pg DNA/20 mg of hair, with an average of 100 pg/mg of hair for the 48 individuals tested. Each symbol represents an individual sample.

Figure 2 depicts the allelic distribution for APO-E 112, APO-E 158, and C825T. Hair shaft DNA samples from 48 representative individuals were genotyped using molecular beacon hybridization. Each square represents the genotype of an independent hair DNA sample as shown in the sample panel: TT (shaded square), TC (black square), or CC (white square).

Figures 3A-3B depict the confirmation of the molecular beacon analysis using direct sequencing and PCR-RFLP analysis. Figure 3 A depicts the nucleotide sequencing of APO-E 158 on 3 representative hair DNA specimens. The SNPs are underlined. Figure 3B depicts PCR-RFLP analysis of C825T on 3 hair shaft DNA samples. PCR products were digested by BseDl to produce different length DNA fragments. BseOl cuts at 5'-CΨCNNGG-3' to yield 2 bands of 33 and 39 bp for the C allele and a single (uncut) 72 bp band for the T allele. Three bands (33, 39 and 72 bp) are present in a heterozygous sample. The 33 and 39 bp bands are very close and appear as a single band.

Detailed Description of the Invention

The present invention is based, at least in part, on the discovery of a method for performing genetic analysis using the DNA isolated from hair shafts, rather than hair roots. The methods of the invention provide a reliable, reproducible method for isolating genomic DNA from the hair shaft in sufficient quantities to perform genetic analysis. The combination of the use of hair-shaft DNA with a highly-sensitive genotyping assay provides a method for performing genetic analysis that is a significant improvement over the prior art.

The methods of the present invention have several major advantages over the prior art, particularly in the areas of population-based genetic screening of humans and in forensic science. The methods of the invention may also be useful in the areas of genotype analysis of laboratory research animals (e.g., mice, rats, monkeys, chimpanzees, rabbits, cats, and dogs) and in livestock (e.g., cows, horses, pigs, goats, and sheep). Hair samples are far more convenient to isolate than tissue (e.g., mucosal swabs), blood, or other body fluid samples. In many cases (e.g., crime scenes), hair may the only type of sample available from which to isolate DNA from suspects and/or victims. Additionally, the methods of the present invention require only the hair shaft, and not the hair root. Therefore, the hair can be isolated simply by cutting the hair, rather than plucking it at the root, which can be moderately invasive, and in many cases, painful. The use of hair shafts as the source of DNA also has advantages in that hair samples are highly stable at room temperature for long periods of time (Sasaki, M. et al. (1997) Forensic Sci. Int. 90:65-75), unlike tissue or fluid samples, which must either be processed soon after isolation, and/or refrigerated or frozen. Accordingly, hair samples can be isolated and/or collected at any geographic location and then mailed at room temperature to the site where the genetic testing is to take place, eliminating the need for personal visits or direct contact with the testing laboratory. Hair samples found at crime scenes can be used for testing even when they are found after long periods of time. In the cases of animals, including laboratory research animals and livestock, genotype analysis using hair shaft DNA provides a totally non-invasive, pain-free method for isolating DNA, eliminating the need for, e.g., isolating DNA from blood, tail-snips, or ear-punches. Another advantage of the methods described herein is that only a small amount of genomic DNA (~ 40 pg) is required for the highly sensitive genotyping assay, thus minimizing the amount of DNA needed for each single analysis and maximizing the information (e.g., number of markers that can be tested) gained from each sample. This is especially important because the amount of hair DNA is generally very small and may not be sufficient for multiple analyses using the conventional prior-art methods. In one specific embodiment, the use of the single-nucleotide polymoφhism (SNP) genotyping method described herein is performed in a single step and can be used for high throughput analysis. A total of 60 different DNA samples can be simultaneously analyzed in one 384-well plate within 4 hours. Finally, the methods described herein are cost-effective and reliable, and may minimizes the non-compliance of subjects in providing samples for DNA isolation.

Isolation of DNA from hair shafts

As described above, the methods of the invention include the isolation of genomic DNA from hair shafts, e.g., hair shafts that do not include the hair root (e.g., the region at the tip of the hair shaft closest to the skin, which includes live follicle cells). Preferably, the methods of the invention utilize cephalic (head) hair, but pubic, body, and/or any other types of hair may also be used. The methods of the invention may use all or a portion of a single hair, or portions of multiple hairs. As used herein, a "portion" of a hair shaft includes a length of the hair shaft that is shorter than the full strand of hair. The methods of the invention also may use combinations and/or mixtures of more than one type of hair from the same subject. The subject is preferably a mammal, most preferably a human, e.g., a human undergoing genetic analysis. The subject may also be animal used in laboratory research (e.g., a mouse, rat, monkey, chimpanzee, rabbit, cat, or dog), a livestock animal (e.g., a pig, goat, sheep, cow, or horse), or any other animal with hair.

Hair may be isolated from the subject by any convenient means. In a preferred embodiment, the hair is cut using a pair of scissors. In other embodiments, the hair may be cut by another other convenient means (e.g., a knife, a razor blade, or other instrument which can cut hair). The hair itself, as well as the instrument used to cut the hair, should be cleaned prior to cutting in order to minimize contamination from other sources of DNA (e.g., hair or skin cells from people or animals who are not the subject to be tested). The hair may also be isolated by pulling it such that it breaks, or by any other means which severs the hair shaft from the subject. After isolation, the hair may be stored in any convenient container, e.g., a tube, bag, box, or envelope, and it may be stored at room temperature, or if desired, it may be refrigerated or frozen.

After isolation of the hair from the subject, the DNA is isolated from the hair using a high-yield DNA isolation method. Preferably, the method includes contacting the hair with an agent (e.g., proteinase K) capable of breaking down the large amounts of keratin protein in the hair. For example, there are many kits known in the art for isolating DNA from tissues. While these kits may not be recommended for use with hair, the discoveries presented herein show that these kits may indeed be used to isolate sufficient quantities of genomic DNA from hair. In a preferred embodiment, the methods of the invention comprise incubating the hair samples with proteinase K to break down and dissolve the hair, and then isolating the DNA from the non-DNA contaminants using a silica-gel membrane, as described in the Examples section herein. Preferably, this method is performed using a DNeasy™ tissue kit (Qiagen, Inc., Valencia, CA), as described herein. Other DNA isolation kits that may be used in the methods of the invention include the PicoPure™ DNA extraction kit (Arcturus, Mountain View, CA), as well as kits from Promega (Madison, WI) and Invitrogen (Carlsbad, CA).

Specific protocols for using any of these kits may be found in instruction manuals of the kits, and are further known to those of skill in the art. The methods of the invention may also use any DNA isolation method which can isolate genomic DNA from hair in quantities sufficient for performing the genotyping assays described herein (e.g., about 40 pg per assay). As described below in the Examples section, the average concentration of genomic

DNA in hair shafts is about 100 pg/mg hair. Twenty mg hair is equivalent to about 2.5 meters (250 cm) of a single hair. A single assay (preferably divided up into about six replicate samples) requires about 40 pg of DNA. Accordingly, about 12.5 cm of hair yields about 1 mg of hair and about 100 pg of DNA, enough for 2.5 assays. However, because the hair of different individuals varies in thickness, the actual length required may also vary according to the degree of thickness.

Genetic Analysis

The methods of the invention utilize highly sensitive assays for determining a subject's genotype at a predetermined locus. As used herein, a "locus" or "genetic locus" is a specific position or location on a chromosome. A wide range of genetic loci are polymoφhic within the population, and there exist a wide range of methods and markers for distinguishing between different alleles at any particular, predetermined locus. As used herein, the term "predetermined" refers to a locus that has been chosen for genetic testing in a particular assay. As used interchangeably herein, the terms "genetic testing" and "genotype analysis" include any method of determining a subject's genotype (i.e., which allele(s) they possess) at a particular locus.

In a preferred embodiment, the genetic loci being tested may be amplified prior to testing, e.g., by polymerase chain reaction (PCR), or by any other method known in the art to amplify DNA.

Preferably, the methods of the invention utilize molecular beacons to detect different alleles at a particular locus. See, for example, the Examples section below. Molecular beacons are oligonucleotide probes that can report the presence of specific nucleic acids in homogeneous solutions (Tyagi, S. et al. (1996) Nat. Biotechnol. 14:303-8; Tyagi, S. et al. (1998) Nat. Biotechnol. 16:49-53). They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes, such as in real-time monitoring of polymerase chain reactions in sealed tubes or for the detection of RNAs within living cells. Molecular beacons are haiφin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid. They are designed in such a way that the loop portion of the molecule is a probe sequence complementary to a target nucleic acid molecule. The stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence. A fluorescent moiety is attached to the end of one arm and a quenching moiety is attached to the end of the other arm. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. Since the quencher moiety is a non-fluorescent chromophore and emits the energy that it receives from the fluorophore as heat, the probe is unable to fluoresce. When the probe encounters a target molecule, it forms a hybrid that is longer and more stable than the stem hybrid and its rigidity and length preclude the simultaneous existence of the stem hybrid. Thus, the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem apart, and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence which can be detected. In order to detect multiple targets in the same solution, molecular beacons can be made in many different colors utilizing a broad range of fluorophores. Dabcyl, a non-fluorescent chromophore, serves as the universal quencher for any fluorophore in molecular beacons. Owing to their stem, the recognition of targets by molecular beacons is so specific that single-nucleotide differences can be readily detected.

Preferred markers/assays used in the genetic analysis methods of the invention SNPs (single nucleotide polymoφhisms). Conventional methods such as direct nucleotide sequencing (e.g., using fluorescently-labeled dye terminators) and RFLP (restriction fragment length polymoφhism) analysis may also be used. Additional methods that may be used include: SCAR (sequence characterized amplified region) analysis, SSLP (simple sequence length polymoφhism) analysis, SSR (simple sequence length repeat) analysis, STS (sequence-tagged site) analysis, microsatellite marker analysis, STSM (sequence-tagged microsatellite marker) analysis, minisatellite marker analysis, polymoφhic repeat analysis, RAPD (random amplified polymoφhic DNA) analysis, AFLP (amplified fragment length polymoφhism) analysis, SAMPL (selective amplification of microsatellite polymoφhic loci) analysis, and CAPS (cleaved amplified polymoφhic sequence) analysis. The specific protocols for performing all of these methods are well-known to those of skill in the art.

Kits

In another embodiment, the invention provides kits for determining the genotype of a subject at at least one predetermined locus (e.g., an SNP such as the APO-E 112 polymoφhic site, the APO-E 158 polymoφhic site, or the GNB3 (C825T) polymoφhic site) comprising reagents suitable for isolating at least about 40 pg of genomic DNA from at least a portion of a hair shaft from the subject, at least two nucleic acid primers which specifically amplify the predetermined locus, and at least two molecular beacon molecules which specifically differentiate between two different alleles of the predetermined locus. In further preferred embodiments, the kits of the invention include primers and molecular beacons specific for at least a second or third predetermined locus.

As used herein, the term "reagents suitable for isolating at least about 40 pg of genomic DNA from at least a portion of a hair shaft from the subject" includes reagents and kits described elsewhere herein. In a preferred embodiment, the primers are selected from the group consisting of SEQ

ID NO:l, 2, 5, 6, 9, or 10. h a further preferred embodiment, the molecular beacons are selected from the group consisting of SEQ ID NO:3, 4, 7, 8, 11, or 12.

In another preferred embodiment, the kits of the invention include instructions (e.g., written instructions) describing the methods for isolating genomic DNA from hair shafts, as wells the methods for determining the genotype at the predetermined locus.

Specific Genotyping Assays

The invention also provides methods for determining the genotypes of individuals at polymoφhic loci in the APO-E and G-protein beta 3 subunit (GNB3) C825T genes. These loci well-known association with human diseases. Certain APO-E genotypes correlate with the risk of developing cardiovascular disease, late onset of Alzheimer's disease (Smith, J.D. (2000) Ann. Med. 32:118-27; Martin, E.R. et al. (2000) Am. J. Hum. Genet. 67:383-94), and sleep-disordered breathing in adults (Kadetani, H. et al. (2001) JAMA 285:2888-90). The genotypes of the C825T polymoφhism of GNB3 correlate- with risk for essential hypertension, obesity and diabetic nephropathy (Benjafield, AN. et al. (1998) Hypertension 32:1094-7; Siffert, W. et al. (1998) Nat. Genet. 18:45-8; Siffert, W. et al. (1999) J. Am. Soc. Nephrol. 10:1921-30; Siffert, W. (2000) Nephrol. Dial. Transplant. 15:1298-306).

The assays for determining the genotypes of individuals at the APO-E and G-protein beta 3 subunit (GΝB3) C825T loci utilize SNP markers and molecular beacon technology, as described in the examples section below.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the sequence listing and the figures, are incoφorated herein by reference.

EXAMPLES

Genomic DNA Purification and Quantitation from Hair Shafts

Cephalic hair shaft samples were collected from 48 anonymous volunteers (40 males and 8 females, ranging in age from 15 to 66 years old) using clean cutting instruments with appropriate consent (Chi-Met Medical Center, Tainan, Taiwan).

DNA was purified from the hair shafts using a DNeasy™ tissue kit (Qiagen, Inc., Valencia, CA), according to the manufacturer's instructions, with slight modifications as described below. Briefly, approximately 20 mg of hair shafts (equivalent to a total length of 2.5 meters of a single hair) were minced and then incubated with 500 μl ATL buffer and 50 μl proteinase K (provided by the kit) at 55°C overnight. The supernatants were transferred to new tubes containing 550 μl AL buffer. After vortexing and incubating at 70°C for 10 minutes, 550 μl of 100% ethanol was added. The samples were subsequently applied to mini-spin columns containing silica-gel membranes, and the DNA was washed and eluted according to the manufacturer's instructions. The DNA was quantified using the PicoGreen® dsDNA quantitation kit (Catalog No. P-l 1496, Molecular Probes, Inc., Eugene, OR) according to the manufacturer's instructions. The fluorescence intensity was measured using a FLUOstar Galaxy fluorescence microplate reader (BMG labtechnologies Inc., Durham, NC). The results were expressed as the average from 6 independent measurements.

Analysis of Genotypes by Single Nucleotide Polymorphism (SNP) PCR Analysis APO-E and G-protein beta 3 subunit (GNB3) C825T loci were selected as the examples in this study because of their well-known association with human diseases. Certain APO-E genotypes correlate with the risk of developing cardiovascular disease, late onset of Alzheimer's disease (Smith, J.D. (2000) Ann. Med. 32:118-27; Martin, E.R. et al. (2000) Am. J. Hum. Genet. 67:383-94), and sleep-disordered breathing in adults (Kadetani, H. et al. (2001) JAMA 285:2888-90). The genotypes of the C825T polymoφhism of GNB3 correlate- with risk for essential hypertension, obesity and diabetic nephropathy (Benjafield, AN. et al. (1998) Hypertension 32:1094-7; Siffert, W. et al. (1998) Nat. Genet. 18:45-8; Siffert, W. et al. (1999) J. Am. Soc. Nephrol. 10:1921-30; Siffert, W. (2000) Nephrol. Dial. Transplant. 15:1298-306). The principles and examples of molecular beacons in allelic determination have been described in previous reports (Tyagi, S. et al. (1996) Nat. Biotechnol. 14:303-8; Tyagi, S. et al. (1998) Nat. Biotechnol. 16:49-53; Shih, I.M. et al. (2001) Cancer Res. 61:818-72). The sequences of the primers and molecular beacons for the three SΝPs tested, APO-E 112, APO- E 158, and GΝB3 (C825T), are as follows:

APO-E 112

Forward Primer: 5'-CGGGCACGGCTGTCCAAG-3' (SEQ TD NO:l) Reverse Primer: 5'-GCATGGCCTGCACCTCGC-3' (SEQ ID NO:2) molecular beacon (Red): 5 '-CACGGACGTGTGCGGCCGCCCGTG-3 ' (SEQ ID NO:3) molecular beacon (Green): 5'-CACGGACGTGCGCGGCCGCCCGTG-3' (SEQ ID NO:4) (The location of the SNP in the molecular beacons is underlined.)

APO-E 158 Forward Primer: 5'-AGGAGCTGCGGGTGCCCC-3' (SEQ JD NO:5) Reverse Primer: 5'-GCCCCGGCCTGGTACACT-3' (SEQ ID NO:6) molecular beacon (Red): 5'-CACGCCTGCAGAAGCGCCTGGCCGTG-3' (SEQ ID NO:7) molecular beacon (Green): 5'-CACGCCTGCAGAAGTGCCTGGCCGTG-3' (SEQ ID NO:8) (The location of the SNP in the molecular beacons is underlined.)

C825T

Forward Primer: 5'-CTCCCACGAGAGCATCATC-3' (SEQ ID NO:9) Reverse Primer: 5'-CCGCCTACTATTCGCTGG-3' (SEQ TD NO: 10) molecular beacon (Red): 5'-CACGATCACGTCCGTGGCCTTCCGTG-3' (SEQ 3D NO: 11) molecular beacon (Green): 5'-CACGATCACGTCTGTGGCCTTCCGTG-3' (SEQ ID NO: 12) (The location of the SNP in the molecular beacons is underlined.)

Both, forward and reverse primers were designed for each SNP, allowing the amplification of approximately 100 bp PCR products. Each hair shaft DNA sample (40 pg) was distributed to 6 wells in a 384- well plate. In addition to all essential reagents, the PCR cocktail contained a pair of molecular beacons labeled with either fluorescein (green fluorescence) or HEX (red fluorescence) that hybridized with the allele harboring the specific SNP (Gene Link, Thornwood, NY and Operon Technologies, Inc., CA). An excess of the reverse primer allowed generation of single-stranded DNA complementary to the molecular beacon. PCR was performed in a single step with the following protocol: 94°C (1 m); 4 cycles of 94°C (15 s), 64°C (15 s), 70°C (15 s); 4 cycles of 94°C (15 s), 61°C (15 s), 70°C (15 s); 4 cycles of 94°C (15 s), 58°C (15 s), 70°C (15 s); 60 cycles of 94°C (15 s), 55°C (15 s), 70°C (15 s); 94°C (1 m) and 60°C (5 m). The fluorescence intensity in each well was then measured by a Galaxy FLUOstar fluorometer (BMG Lab Technologies, Durham, NC), the ratio of fluorescein/HEX fluorescence intensity was determined from each well, and the average from 6 repeats on each sample was determined. The data were converted into genotypes by a computer program.

PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis

The PCR product that contained C825T was digested with BseDl (Fermentas, Hanover, MD) at 55°C overnight (as described in Benjafield, AN. et al. (1998) Hypertension 32:1094-7 and Siffert, W. et al. (1998) Nat. Genet. 18:45-8) and was elecrrophoresed on a 10% polyacrylamide gel (Νovex®, Invitrogen Inc., Carlsbad, CA). The different alleles were discriminated by the length of PCR product BseDl digestion.

Nucleotide Sequence Analysis

DΝA from the wells of the PCR plates was purified using a MinElute™ PCR purification kit (Qiagen, Inc., Valencia, CA) and was subjected to nucleotide sequencing. The primers used for sequencing were the forward primers listed above. Sequencing was performed using fluorescently labeled Applied Biosystems Big Dye terminators and an Applied Biosystems 377 automated sequencer (Applied Biosystems, Foster City, CA).

Statistical Analysis

Chi-square comparison of proportions was used to analyze the significance of the difference in allelic ratio among different populations, p values less than 0.05 were considered statistically significant.

Results

The concentration of hair shaft DΝA ranged from 0.02 to 20 ng/20 mg of hair in the 48 individuals tested, with an average of 100 pg/mg of hair (Figure 1). The hair DΝA concentration was similar between male and female hair (p > 0.1 ).

The hair DΝA was used for APO-E and GΝB3 (C825T) genotyping in which the loci contain SNPs that were reliably distinguished by a pair of molecular beacons. Using molecular beacon hybridization, the different genotypes were assigned to individuals based an the fluorescence intensity ratio of the two molecular beacons (Figure 2). The genotypes determined by the molecular beacons were confirmed by direct nucleotide sequencing or PCR-RFLP analysis in 40 representative individuals (Figure 3). The genotypes determined using the hair shaft DNA were additionally confirmed by testing lymphocyte DNA isolated from the same individuals. The sensitivity of the method was assessed by analyzing different amounts of hair shaft DNA. The results demonstrated that as little as 40 pg of DNA gave reproducible genotyping results. Tables I, II, and III below summarize the genotype results of genotypes of APO-E and GNB3 (C825T) in the Taiwanese population.

Table I: APC )-E Genotype Pattern

Sample # Codon Genotype Pattern

112 158 allele 1 / allele 2

34* TT TT E2 (τ¹¹²T¹⁵⁸) / E2 (τ¹¹²T¹⁵⁸)

1 TT CC E3 (T¹¹²C¹⁵⁸) / E3 (T¹¹²C¹⁵⁸)

3 CC CC E4 (C¹¹²C¹⁵⁸) / E4 (C¹¹²C¹⁵⁸)

6 TT TC E2 (τ¹¹²τ¹⁵⁸) / E3 (T¹¹²C¹⁵⁸)

12 TC CC E3 (T¹¹²C¹⁵⁸) / E4 (C¹¹²C¹⁵⁸) *Representative samples from Figure 2

Table II: APO-E Genotype Frequency

Population n Genotype (%) Allele (%)

E2/E2 E2/E3 E3/E3 E2/E4 E3/E4 E4/E4 ε2 ε3 ε4

Taiwanese 48 2.1 14.6 68.8 0.0 12.5 2.1 9.4 82.3 8.3^C

Chinese^a 141 1.4 12.1 70.9 0.0 14.9 0.7 7.4 84.4 8.2^C

European 590 0.6 9.3 57.3 1.9 27.3 3.4 6.4 75.6 16.0^d ^a Evans, A.E. et al. (1993) Hum. Genet. 92:191-7 Hallman, D.M. et al. (1991) Am. J. Hum. Genet. 49:338-49 ^c not significant ^άp < 0.03 Table III: C825T Genotype Frequency

Population n Genotype (%)

TT TC CC

Taiwanese 48 23.0^b 41.0 36.0

Chinese^a 960 22.4 50.6 27.0

European^a 277 10.0^C 43.7 46.2

" Siffert, W. et al. (1999) J. Am. Soc. Nephrol. 10:1921-30 ^b not significant ^cp < 0.001

For APO-E genotypes, there were two polymoφhic sites (codons 112 and 158) in one haplotype, thus resulting in three distinct allelic types (E2, E3 and E4) (Table I) (Zannis, V.I. et al. (1981) Am. J. Hum. Genet. 33:11-24; Utermann, G. et al. (1980) Am. J. Hum. Genet. 32:339-47). When compared with the genotypes of Chinese (Mainlander) and Europeans (Icelander, Finnish and Hungarian) (Siffert, W. et al. (1999) J. Am. Soc. Nephrol. 10:1921- 30; Hallman, D.M. et al. (1991) Am. J. Hum. Genet. 49:338-49), the percentage of individuals carrying the APQ-E risk allele (E4) for Alzheimer's disease was similar to that in Chinese but significantly lower than that in Europeans (p = 0.027) (Tables II and III). For GNB3 (C825T), the percentage of Taiwanese with the risk allele (TT) was also similar to Chinese but was significantly higher than that in Europeans (p = 0.002).

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

I . A method of determining the genotype of a subject at at least one predetermined locus comprising: a) isolating at least about 40 pg of genomic DNA from at least a portion of a hair shaft from the subject; and b) testing said genomic DNA to determine the genotype of the subject at the predetermined locus.

2. The method of claim 1, wherein the hair shaft does not comprise the hair root.

3. The method of any of claims 1 -2, wherein the hair shaft is a cephalic hair shaft.

4. The method of any of claims 1 -2, wherein the hair shaft is selected from the group consisting of a pubic hair shaft and a body hair shaft.

5. The method of any of claims 1 -4, wherein the subj ect is a mammal.

6. The method of claim 5, wherein the subject is a human.

7. The method of any of claims 1-6, wherein the predetermined locus is amplified prior to the testing.

8. The method of claim 7, wherein the predetermined locus is amplified using the polymerase chain reaction (PCR).

9. The method of any of claims 1-8, wherein the predetermined locus is a single- nucleotide polymoφhism (SNP).

10. The method of any of claims 1 -9, wherein the predetermined locus is the

APO-E 112 polymoφhic site.

I I . The method of any of claims 1 -9, wherein the predetermined locus is the APO-E 158 polymoφhic site.

12. The method of any of claims 1 -9, wherein the predetermined locus is the GNB3 (C825T) polymoφhic site.

13. The method of any of claims 1-12, further comprising determining the genotype of the subject at at least a second locus.

14. The method of claim 13, wherein the second locus is selected from the group consisting of the APO-E 112 polymoφhic site, the APO-E 158 polymoφhic site, and the GNB3 (C825T) polymoφhic site.

15. The method of any of claims 13-14, further comprising determining the genotype of the subject at at least a third locus.

16. The method of claim 15, wherein the third locus is selected from the group consisting of the APO-E 112 polymoφhic site, the APO-E 158 polymoφhic site, and the GNB3 (C825T) polymoφhic site.

17. The method of any of claims 1-16, wherein the testing comprises the use of at least two molecular beacons capable of differentiating between the different alleles of the predetermined locus.

18. The method of any of claims 1-16, wherein the testing comprises direct sequencing of the predetermined locus.

19. The method of any of claims 1-16, wherein the testing comprises RFLP (restriction fragment length polymoφhism) analysis.

20. The method of any of claims 1-16, wherein the testing comprises a method selected from the group consisting of: SCAR (sequence characterized amplified region) analysis, SSLP (simple sequence length polymoφhism) analysis, SSR (simple sequence length repeat) analysis, STS (sequence-tagged site) analysis, microsatellite marker analysis, STSM (sequence-tagged microsatellite marker) analysis, minisatellite marker analysis, polymoφhic repeat analysis, RAPD (random amplified polymoφhic DNA) analysis, AFLP (amplified fragment length polymoφhism) analysis, SAMJPJL (selective amplification o± microsatellite polymoφhic loci) analysis, and CAPS (cleaved amplified polymoφhic sequence) analysis.

21. The method of any of claims 1 -20, wherein the DNA is isolated using a method comprising treating the hair shaft with proteinase K.

22. The method of any of claims 1-21, wherein the DNA is isolated using a method comprising selectively binding the DNA to a silica-gel membrane that does not substantially bind non-DNA contaminants.

23. The method of any of claims 1 -22, wherein the portion of a hair shaft comprises about 20 mg of hair.

24. A kit for determining the genotype of a subject at at least one predetermined locus comprising: a) reagents suitable for isolating at least about 40 pg of genomic DNA from at least a portion of a hair shaft from the subject; b) at least two nucleic acid primers which specifically amplify the predetermined locus; c) at least two molecular beacon molecules which specifically differentiate between two different alleles of the predetermined locus; and d) instructions for use.

25. The kit of claim 24, wherein the predetermined locus is amplified using the polymerase chain reaction (PCR).

26. The kit of any of claims 24-25, wherein the predetermined locus is a single- nucleotide polymoφhism (SNP).

27. The kit of any of claims 24-26, wherein the predetermined locus is the APO-E 112 polymoφhic site.

28. The kit of any of claims 24-26, wherein tήe predetermined locus is the APU-E 158 polymoφhic site.

29. The kit of any of claims 24-26, wherein the predetermined locus is the GNB3 (C825T) polymoφhic site.

30. The kit of any of claims 24-29, further comprising: a) at least two nucleic acid primers which specifically amplify a second predetermined locus; and b) at least two molecular beacon molecules which specifically differentiate between two different alleles of the second predetermined locus.

31. The kit of claim 30, wherein the second locus is selected from the group consisting of the APO-E 112 polymoφhic site, the APO-E 158 polymoφhic site, and the GNB3 (C825T) polymoφhic site.

32. The kit of any of claims 30-31, further comprising: a) at least two nucleic acid primers which specifically amplify a third predetermined locus; and b) at least two molecular beacon molecules which specifically differentiate between two different alleles of the third predetermined locus

33. The kit of claim 32, wherein the third locus is selected from the group consisting of the APO-E 112 polymoφhic site, the APO-E 158 polymoφhic site, and the GNB3 (C825T) polymoφhic site.

34. The kit of any of claims 24-33, wherein the primers are selected from the group consisting of SEQ ID NO:l, 2, 5, 6, 9, or 10.

35. The kit of any of claims 24-34, wherein the molecular beacons are selected from the group consisting of SEQ D NO:3, 4, 7, 8, 11, or 12.