Abstract:
Biotinidase deficiency is detected by determining the activity of the biotinidase enzyme utilizing a newborn dried blood spot and calorimetric end point analysis. The four mutations most commonly associated with complete biotinidase deficiency are G98: d7i3, Q456H, R538C, and the double mutation D444H:A171T. Partial biotinidase deficiency is almost universally attributed to the D444H mutation. To more effectively distinguish between profound and partial biotinidase deficiency, a panel of assays utilizing real time PCR and melting curve analysis is developed to detect those mutations listed above. In newborn screening for biotinidase deficiency, the analysis of common mutations is useful to distinguish between partial and complete enzyme deficiency. Combining biotinidase enzyme analysis with genotypic data also increases the sensitivity of screening for biotinidase deficiency and provides information useful to clinicians earlier than would otherwise be possible.

Description:
SPECIFIC REFERENCE  
       [0001]    The present application claims benefit of provisional application Ser. No. 60/400264, filed Aug. 1, 2002. 
     
    
     
       BACKGROUND  
         [0002]    1. Description of the Related Art  
           [0003]    Biotinidase deficiency is an autosomal recessive metabolic disorder occurring in 1:80,000 live births. Those affected by biotinidase deficiency exhibit irreversible neurological problems, seizures, developmental delays, hypotonia, ataxia, cutaneous and other symptoms. Symptoms are preventable and in some cases reversible through oral biotin supplementation. Prospective newborn screening for biotinidase deficiency is, therefore, performed in much of the USA and in numerous other countries. Biotinidase deficiency results from mutations in the biotinidase gene and depending upon the nature of the mutation(s), the enzyme deficiency may be either complete or partial. Mean biotinidase activity is 7.1 nmol/min/ml serum in normal newborns. Those affected with complete biotinidase deficiency have enzymes that produce &lt;10% of mean normal activity, while those affected with partial deficiency have enzymes that produce 10-30% of normal activity.  
           [0004]    In newborn screening laboratories, assaying for biotinidase deficiency is performed using an extract of whole blood derived from the universally collected newborn dried blood spot (DBS). Whole blood extracted from a DBS specimen is an effective sample from which to assay for biotinidase activity, but not as precise as results obtained with serum. Activity of the biotinidase enzyme may be adversely affected if the DBS specimen is mishandled. DBS specimens that are incompletely dried, exposed to moisture after drying, or exposed to heat may exhibit reduced biotinidase activity. Inconclusive or ambiguous results in screening for biotinidase deficiency are therefore often attributable to errors in sample collection and processing prior to their arrival in the screening laboratory. Another difficulty experienced in newborn screening for biotinidase deficiency involves differentiating between a complete and a partial enzyme deficiency. To aid in distinguishing between complete and partial biotinidase deficiency and subsequently increasing the sensitivity and specificity of screening for biotinidase deficiency, mutational analysis has been employed.  
           [0005]    In the United States, the following 5 mutations are the most frequently observed in patients with biotinidase deficiency: Q456H, R538C, G98:d7i3, D444H, and the double mutation D444H:A171T. Q456H, R538C, and G98:d7i3 are associated with complete biotinidase deficiency. The D444H mutation has a carrier rate of 3.9% in the general population and causes partial enzyme deficiency. This high frequency in the general population combined with its causing a partial enzyme deficiency makes the D444H mutation similar to the Duarte D2 N314D variant in galactosemia. Interestingly, when D444H is in cis with the A171T mutation, the combined deleterious effects of both mutations result in an allele causing complete enzyme deficiency. The D444H:A171T double mutation is commonly observed in biotinidase deficient children that are ascertained by newborn screening. Using Light Cycler technology and paired hybridization probes, assays were designed to detect these 5 mutations frequently observed in biotinidase deficiency. Reported here are the assay procedures to detect these 5 commonly observed biotinidase mutations together with results from the analysis of specimens identified as presumptively positive in our prospective screening program for biotinidase deficiency. This use of mutational analysis may supplement the biochemical screening results and increases the specificity of newborn screening for biotinidase deficiency.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0006]    [0006]FIG. 1 shows the melting curve results after analysis of the R538C biotinidase mutation.  
         [0007]    [0007]FIG. 2 shows the melting curve results after analysis of the Q456H biotinidase mutation.  
         [0008]    [0008]FIG. 3 shows the melting curve results after analysis of the G98:d7i3 biotinidase mutation.  
         [0009]    [0009]FIG. 4 shows the melting curve results after analysis of the D444H biotinidase mutation.  
         [0010]    [0010]FIG. 5 shows the melting curve results after analysis of the A171T biotinidase mutation. 
     
    
     SUMMARY OF THE INVENTION  
       [0011]    The present invention relates to newborn screening for biotinidase deficiency using assays involving PCR amplification and Light Cycler platform technologies. 5 common mutations including G98:d7i3, Q456H, R538C, A171T, and D444H are now capable of being detected by a comparison of the hybrid melting temperatures with patient specimens.  
         [0012]    Accordingly, what is provided is a method for detecting biotinidase deficiency for newborn screening, comprising amplifying a DNA strand from a specimen to thereby form an amplification product; wherein the amplification product is specific for detecting a mutation frequently observed in patients with biotinidase deficiency; allowing a pair of labeled probes to hybridize to one strand of the amplification product, wherein a detection probe is adapted to match to a sequence that may include the mutation, and an anchor probe hybridizes to an adjacent sequence, thereby forming hybrids; allowing fluorescence resonance energy transfer to occur between a donor fluorophore and an acceptor fluorophore of each hybrid, wherein an excitation wavelength of the donor fluorophore and a fluorescence of the acceptor fluorophore is acquired; and, generating a melting curve having peaks indicative of the melting temperature (Tm) of each hybrid.  
       DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0013]    Specimens and DNA Preparation  
         [0014]    Biotinidase deficient specimens, ascertained through either routine prospective newborn screening or high-risk screening, were retrieved from a specimen archive. Specimens retrieved from archival storage were utilized in assay development and the retrospective study. DNA was isolated from DBS specimens and 80-130 ng was utilized as template in each reaction. Specimens, characterized as homozygous for Q456H, were provided by the Department of Pediatrics, University Hospital Vienna, Vienna, Austria.  
         [0015]    Prospective Newborn Screening for Biotinidase Deficiency  
         [0016]    DBS specimens are submitted by hospitals. Analysis of biotinidase activity is routinely performed using the Astoria Pacific Continuous Flow Analyzer and the Astoria Pacific SPOTCHECK biotinidase enzyme assay reagents. These reagents, to assay biotinidase activity in DBS specimens, are based upon those methods described by Wolf et al. in a screening method for biotinidase deficiency in newborns,  Clin. Chem . (1):125-127, 1984. Samples demonstrating biotinidase activity below the critical cut-off level of 20.0 eru (enzyme response units) are selected for genotype analysis.  
         [0017]    Polymerase Chain Reaction and Hybridization Probes  
         [0018]    Sequences of the human biotinidase gene (Genbank accession numbers NM00060 (SEQ. ID NO: 1), AF018630 (SEQ. ID NO: 2), AF18631 (SEQ. ID NO: 3) were the basis from which primers and probes were designed. Primer Premier 5 (Premier Biosoft, Palo Alto, Calif.) and Tm Utility 1.5 IT (Idaho Technology, Salt Lake City, Utah) software were utilized as aids to design primers for polymerase chain reaction (PCR) and hybridization probes to detect mutations. Primers and fluorescent labeled probes may also be obtained from either Operon Technology (Alameda, Calif.) or Idaho Technology (Salt Lake City, Utah). PCR reaction buffers may be obtained from Idaho Technology (Salt Lake City, Utah.). Amplification reactions utilize 1× PCR buffer, 2 mM MgCl 2 , 200 μM dNTPs (Roche, Manheim, Germany), and 0.6U Klen taq (AB Peptides, St. Louis, Mo.) in a complex with TaqStart antibody (ClonTech, Palo Alto, Calif.). Preparing a complex between the polymerase and TaqStart antibody is performed according to manufacturer&#39;s instructions. The sequence of individual primers, the sequence of fluorescent hybridization probes, and the concentration at which each is used are found in Tables 1-3 as follows:  
                                                                         TABLE 1                       Assay   Forward Primer   conc.   Seq. ID   Reverse Primer   conc.   Seq. ID                                G98:d7i3   GCCCCATTACATTCCAGATTTG   0.5   4   CTCATACACGGCAGCCACAT   1.0   9                   Q456H   GCCCACCTTATCCAAAGAGC   0.5   5   GGTGTCGAAGCCAAGACCC   1.0   10               R538C   GCTTGGCTGGGAGAATGACC   0.5   6   CTTGTAGCCTGTGGAAGTGC   1.0   11               D444H   GGGGAAAGGAAGGCTATCTC   1.0   7   ACAGGTGTCGAAGCCAAGAC   0.5   12               A171T   CTCCAGCGCCTGAGTTGTAT   0.13   8   TCCATTATTGCTGAACACGAC   0.25   13                  
 
         [0019]    [0019]                                         TABLE 2                       Assay   Anchor Probe   Seq. ID No.                                G98:d7i3   TGGTCTGCATTATGTCTGGAGCCAGAAGTA-fitc   14                   Q456H   TTTGATGGGCTTCACACAGTACATGGCACT-fitc   15               R538C   LCred640-AGGGACTAGGAAAAGTGTGTGGTCTGTGG-P   16               D444H   LCred640-AGGGCATACAGCTCTTTGGATAAGGTGGGC   17               A171T   LCred640-AGGAGCCTTGTCATAGCAGTGACCCAAGGT-P   18                    
         [0020]    [0020]                                             TABLE 3                               Seq. ID           Assay   Detection Probe   No.                                G98:d7i3   LCred640-GCTTGCTCTTTTCCTCTGCG-P   19                   Q456H   LCred640-ACTACATCCACGTGTGTGCCC-P   20               R538C   CTCTATGGGCGCTTGTATGA-fitc   21               D444H   TGAAGCCCATCAAAGACCCC-fitc   22               A17IT   TGGTGACCAATCTTGGGACA-fitc   23                            
         [0021]    Ten microliter PCR reactions were performed in capillary tubes using a Roche Light Cycler (Manheim, Germany). Temperature cycling conditions for PCR utilizes a modified 2-step thermal cycling scheme. Specimens are ramped to 94° C. at 20°/second and held there for 0.0 seconds to denature the DNA strands. Temperature then ramps at 20°/second to 58° C. and holds at this temperature for 15 seconds at which time primers anneal and polymerization of new DNA begins. Polymerization is completed while ramping from 58° C. to 72° C. at 1.0°/second. The slow ramp speed allows polymerization to proceed, thus negating the necessity of a hold time at 72° C. Thermal cycling is repeated for 45 cycles. All amplifications are preferably performed in an asymmetric manner. Asymmetric amplifications for G98:d7i3, Q456H, R538C, and A171T assays enrich the antisense strand of the amplicon while the asymmetric amplification in the D444H assay enriches the sense strand of the amplicon. Asymmetry produces an excess of the DNA strand to which the hybridization probes will bind in the analysis phase of the assay.  
         [0022]    Hybridization Probes and Genotyping Analysis  
         [0023]    Genotyping is performed using paired hybridization probes, where each assay has a detection probe and an anchor probe. Probes for the G98:d7i3, Q456H, R538C, and A171T assays hybridize to the antisense strand of the amplicon while the probes for D444H assay hybridize to the sense strand of the amplicon. The detection probe hybridizes with a region of the amplicon that includes the mutation, while the anchor probe hybridizes with a region adjacent to the detection probe. When both probes are hybridized, there is a 1 base gap between the anchor and detection probes. For each set of hybridization probes, one is conjugated on the 3&#39;end with fitc while the second is conjugated on the 5′ end with LC red640. The probe which is 5′ conjugated with LC red640 is also 3′ phosphorylated to prevent extension by taq DNA polymerase. When both probes are hybridized with the amplicon, the fluorescent moieties are brought into close proximity, and this proximity allows fluorescence resonance energy transfer to occur between the donor fluorophore (fitc) and the acceptor fluorophore (LC red640). Anchor probes have a Tm that is at least 15% higher than the corresponding detection probe, which allows the anchor probe to remain hybridized during the melting transition of the detection probe that occurs during the analysis phase of the assay.  
         [0024]    After completing the thermal cycling program, the Light Cycler proceeds seamlessly to the analysis program. The analysis program ramps to 94° C. at 20°/second and after reaching 94° C., immediately begins to ramp at 1°/second to 35° C. Upon reaching 35° C. the temperature ramps upward at 0.1°/second to 76° C. During the entire analysis program, the excitation wavelength of fitc is provided and the fluorescence of LC red640 is continuously acquired. Melting curves are generated by plotting the fluorescence of LC red640 against temperature during the 35°-76° upward temperature ramp. Melting peaks are generated computationally by calculating the −dF/dT of the melting curve which is then plotted against temperature.  
       RESULT EXAMPLES  
       [0025]    Detecting Frequently Observed Mutations in the Biotinidase Gene.  
         [0026]    [0026]FIGS. 1-5 display analysis of individual biotinidase mutations using melting peaks generated with the Light Cycler. FIG. 1 displays the assay results for the R538C mutation and specimens that are homozygous wild type, heterozygous, and no DNA control are analyzed. No specimen that is homozygous for R538C has yet been identified. The remainder of the assays, shown in FIGS. 2-5 display specimens that are homozygous wild type, heterozygous, homozygous for the mutation, and a no DNA control. In the cases of the D444H, G98:d7i3, and R538C assays, the detection probe matches the wild type sequence. Therefore, the high temperature melting peak represents the wild type form of the gene while the low temperature melting peak represents the mutant form of the gene. In the A171T and Q456H assays, the detection probe matches the mutant form of the gene and has a 1 base pair mismatch with the wild type allele. In these assays, the high-temperature melting peak represents the mutant form of the gene while the low-temperature melting peak represents the wild type form of the gene.  
         [0027]    Analysis of Specimens Identified through Newborn Screening as Presumptive Positive for Biotinidase Deficiency.  
         [0028]    Through newborn screening, 49 specimens were identified as presumptively positive for biotinidase deficiency. Of these 49 specimens, 45 were suitable for genotype analysis. In the cases of the 4 specimens that were not analyzed, there was inadequate dried blood remaining on the DBS to obtain a DNA specimen. These 45 specimens were analyzed for the 5 mutations and genotyping results are shown in Table 4 as follows:  
                               TABLE 4                                       Genotype       Spec-               Based Preliminary        imen   Origin   Allele 1   Allele 2   Diagnosis                   BD1   Domestic   D444H/A171T   D444H   Partial Deficiency       BD2   Domestic   Q456H   ND   Incomplete                       Genotype       BD3   Domestic   G98:d7i3   D444H   Partial Deficiency       BD4   Domestic   Q456H   ND   Incomplete                       Genotype       BD5   Domestic   ND   ND   —       BD6   Domestic   G98:d7i3   ND   Incomplete                       Genotype       BD7   Domestic   Q456H   ND   Incomplete                       Genotype       BD8   Domestic   Q456H   ND   Incomplete                       Genotype       BD9   Brazil   ND   ND   —       BD10   Domestic   D444H/AI7IT   R538C   Complete                       Deficiency       BD11   Turkey   D444H   D444H   Partial Deficiency       BD12   Domestic   Q456H   D444H   Partial Deficiency       BD13   Chile   D444H   ND   Putative                       Partial Deficiency       BD14   India   G98:d7i3   G98:d7i3   Complete                       Deficiency       BD15   Mexico   D444H   ND   Putative                       Partial Deficiency       BD16   Chile   ND   ND   —       BD17   Domestic   ND   ND   —       BD18   Domestic   ND   ND   —       BD19   Domestic   D444H   D444H   Partial Deficiency       BD20   Domestic   Q456H   ND   Incomplete                       Genotype       BD21   Domestic   D444H   ND   Putative                       Partial Deficiency       BD22   Brazil   G98:d7i3   ND   Incomplete                       Genotype       BD23   India   G98:d7i3   G98:d7i3   Complete                       Deficiency       BD24   Domestic   D444/A171T   D444H/A171T   Complete                       Deficiency       BD25   Domestic   D444H   ND   Putative                       Partial Deficiency       BD26   Domestic   Q456H   D444H   Partial Deficiency       BD27   Domestic   D444H/A171T   D444H   Partial Deficiency       BD28   Domestic   D444H   D444H   Partial Deficiency       BD29   Domestic   D444H   D444H   Partial Deficiency       BD30   Domestic   D444H   D444H   Partial Deficiency       BD31   Domestic   D444H   D444H   Partial Deficiency       BD32   Domestic   D444H   ND   Putative                       Partial Deficiency       BD33   Brazil   D444H   D444H   Partial Deficiency       BD34   Domestic   Q4565H   ND   Incomplete                       Genotype       BD35   Domestic   R538C   D444H   Partial Deficiency       BD36   Domestic   D444H   ND   Putative                       Partial Deficiency       BD37   Domestic   Q456H   Q456H   Complete                       Deficiency       BD38   Domestic   D444H:A171T   ND   Incomplete                       Genotype       BD39   Domestic   Q456H   D444H   Partial Deficiency       BD40   Domestic   D444H   ND   Putative                       Partial Deficiency       BD41   Domestic   Q456H   ND   Incomplete                       Genotype       BD42   Domestic   D444H   ND   Putative                       Partial Deficiency       BD43   Domestic   Q456H   D444H   Partial Deficiency       BD44   Domestic   R538C   D444H   Partial Deficiency       BD45   Domestic   D444H   D444H   Partial Deficiency                          
 
         [0029]    Thirty-six specimens were of domestic origin and 9 were of foreign origin (see table 4 for the countries of origin). Overall, in 88.8% (40/45) of the specimens at least 1 mutation was identified. For specimens of domestic origin, 91.6% (33/36) contained at least 1 mutation, while 78% of specimens of foreign origin (7/9) contained at least 1 mutation. A complete genotype was obtained from 21 specimens. Seventeen specimens (BD1, BD3, BD11, BD12, BD19, BD26-31, BD33, BD35, BD39, BD43-45) could be assigned a preliminary designation of partial deficiency because they were either homozygous for D444H or were compound heterozygous between D444H and one of the other mutations being assayed for. The genotypes of 5 specimens (BD10, BD14, BD23, BD24, BD37) clearly identified them as complete deficiencies, 2 of which were homozygous for G98:d7i3 (BD14, BD23), one was homozygous for D444H:A171T (BD24), one was homozygous for Q456H (BD37), and one was a compound heterozygote for D444H:A171T and R538C (BD10). Eight additional specimens (BD13, BD15, BD21, BD25, BD32, BD36, BD40, BD42) could be assigned a preliminary designation of partial deficiency owing to the presence of a single copy of the D444H mutation and reduced enzyme activity. Mutations observed in specimens of foreign origin were limited to D444H and G98:d7i3, while all 5 mutations were observed in specimens of domestic origin.  
         [0030]    In newborn screening for biotinidase deficiency, it is frequently difficult to discern if a partial or complete enzyme deficiency has been encountered. In the vast majority of partial deficiencies, the D444H mutation is involved (8). D444H has a carrier frequency of 3.9% in the general United States population and reduces the activity of the biotinidase enzyme by 48-52%. It is noteworthy, that these percent reductions were determined with serum quantitative enzyme analysis, thus the percent enzyme reduction in a DBS derived whole blood specimen could be greater. Partial deficiencies are either homozygous for D444H or compound heterozygotes with D444H and a second mutation. In Table 2, there are seventeen specimens with genotypes identifying them as partial deficiencies. Nine specimens are compound heterozygous with D444H and a second mutation, while eight are homozygous for D444H. Additionally, there are eight other specimens where a single copy of D444H is identified. This is strong evidence that these too are partial deficiencies. It is unlikely that these eight specimens with a single copy of D444H are simple carriers because the enzyme assay was below the critical cut-off and a carrier of D444H would not be expected to produce such low enzyme activity. This suggests that such specimens are likely compound heterozygotes having one copy of D444H and a rare or private mutation in the second copy of the biotinidase gene. In the enzyme assay used in newborn screening, compound heterozygotes containing D444H and a mutation causing a complete deficiency (R538C, G98:d7i3, Q456H, etc) may generate biochemical data effectively mimicking complete biotinidase deficiency. A similar situation is frequently observed in the Beutler assay that is used to measure GALT activity when screening for galactosemia. Compound heterozygotes between the Duarte D2 N314D variant and a classical galactosemia mutation such as Q188R may generate biochemical data suggesting classical galactosemia. Identifying the N314D GALT mutation provides definitive proof that these specimens are not classical galactosemia. In a similar fashion, the D444H mutation is responsible for the vast majority of partial biotinidase deficiencies and therefore identifying this mutation provides strong evidence that a complete enzyme deficiency is not present.  
         [0031]    A complication surrounding the D444H mutation is double mutants. Three double mutations, involving D444H and a second mutation on the same gene, have been described. The commonly observed double mutant, D444H:A171T, that accounts for 17.3% of the mutations in complete biotinidase deficiencies ascertained by newborn screening, is part of this panel. Indeed, specimen BD24 from Table 2 is homozygous for D444H:A171T. Two other double mutations, D444H:F403V and D444H:R157H, have been described, however both are extremely rare. The D444H mutation is very useful to identify partial deficiencies, but the possibility of a rare or unique double mutant resulting in a complete deficiency cannot be dismissed. After newborn screening results are reported, the first clinical visit of a potential biotinidase deficient newborn will involve determining quantitative serum biotinidase activity and possibly confirmatory molecular diagnostic analysis. Quantitative biotinidase analysis is the ultimate diagnostic test to identify biotinidase deficiency and the newborn screening analysis is secondary to these results. Second tier mutation screening is to benefit the newborn screening program and acts as a guide in clinical evaluation. However in certain situations, as are observed in specimens BD10, BD14, BD23, BD24, and BD37, the genotype data unambiguously identifies these specimens as having a complete enzyme deficiency. Such informative results can expedite patient care to get the newborn immediate attention.  
         [0032]    The data shown in Table 4 and discussed above provides evidence to the utility of second tier mutation analysis in newborn screening for biotinidase deficiency. In a high throughput newborn screening laboratory, the most important issue is validity of results, but following closely behind is turn around time. Minimizing turn around time requires that assay platforms be fast, reliable. and easily interpreted within the context of a routine service laboratory. The Light Cycler platform is ideal for the high throughput newborn screening laboratory because all of these criteria are met. Air driven thermal cycling is fast, genotyping with fluorescent hybridization probes is simple because it involves no post-PCR manipulation, and melting peak data is easily interpreted. From isolation of DNA to data interpretation, the 5-mutation panel described here is completed in less than 2 hours. Such rapid analysis assures that second tier molecular data is reported along with the primary biochemical data. An additional benefit is that the close tube format simplifies sample tracking and is favorable for avoiding amplicon contamination in the laboratory. Data files from the Light Cycler are easily stored and may be backed up in an off-site archive rendering them safe from loss. This is an ideal situation for the newborn screening laboratory where large quantities of sensitive clinical data are generated.  
         [0033]    In the example above, mutations that cause biotinidase deficiency were identified. Among specimens of domestic origin identified using the present methodology, the panel of five mutations proved useful in 91.6 % of presumptive positive newborns. Biochemical analysis will remain the primary means by which biotinidase deficiency is detected in both newborn screening and clinical diagnostics. Second tier mutation analysis provides valuable support to biochemical analysis and should be considered as a supplement to the biochemical data by those performing newborn screening for biotinidase deficiency.  
     
       
       
         1 
         
           
             23  
           
           
             1  
             2016  
             DNA  
             Homo sapiens  
           
            1 

gccagctgga gcgttttcgg ggctgtaaag ggagaatggc gcatgcgcat attcagggcg     60 

gaaggcgcgc taagagcaga tttgtggtct gcattatgtc tggagccaga agtaagcttg    120 

ctcttttcct ctgcggctgt tacgtggttg ccctgggagc ccacaccggg gaggagagcg    180 

tggctgacca tcacgaggct gaatattatg tggctgccgt gtatgagcat ccatccatcc    240 

tgagtctgaa ccctctggct ctcatcagcc gccaagaggc cttggagctc atgaaccaga    300 

accttgacat ctatgaacag caagtgatga ctgcagccca aaaggatgta cagattatag    360 

tgtttccaga agatggcatt catggattca actttacaag aacatccatt tatccatttt    420 

tggacttcat gccgtctccc caggtggtca ggtggaaccc atgcctggag cctcaccgct    480 

tcaatgacac agaggtgctc cagcgcctga gttgtatggc catcagggga gatatgttct    540 

tggtggccaa tcttgggaca aaggagcctt gtcatagcag tgacccaagg tgcccaaaag    600 

atgggagata ccagttcaac acaaatgtcg tgttcagcaa taatggaacc cttgttgacc    660 

gctaccgtaa acacaacctc tactttgagg cagcattcga tgttcctctt aaagtggatc    720 

tcatcacctt tgataccccc tttgctggca ggtttggcat cttcacatgc tttgatatat    780 

tgttctttga ccctgccatc agagtcctca gagactacaa ggtgaagcat gttgtgtacc    840 

caactgcctg gatgaaccag ctcccactct tggcagcaat tgagattcag aaagcttttg    900 

ctgttgcctt tggcatcaac gttctggcag ctaatgtcca ccacccagtt ctggggatga    960 

caggaagtgg catacacacc cctctggagt ccttttggta ccatgacatg gaaaatccca   1020 

aaagtcacct tataattgcc caggtggcca aaaatccagt gggtctcatt ggtgcagaga   1080 

atgcaacagg tgaaacggac ccatcccata gtaagttttt aaaaattttg tcaggcgatc   1140 

cgtactgtga gaaggatgct caggaagtcc actgtgatga ggccaccaag tggaacgtga   1200 

atgctcctcc cacatttcac tctgagatga tgtatgacaa tttcaccctg gtccctgtct   1260 

ggggaaagga aggctatctc cacgtctgtt ccaatggcct ctgctgttat ttactttacg   1320 

agaggcccac cttatccaaa gagctgtatg ccctgggggt ctttgatggg cttcacacag   1380 

tacatggcac ttactacatc caagtgtgtg ccctggtcag gtgtgggggt cttggcttcg   1440 

acacctgcgg acaggaaatc acagaggcca cggggatatt tgagtttcac ctgtggggca   1500 

acttcagtac ttcctatatc tttcctttgt ttctgacctc agggatgacc ctagaagtcc   1560 

ctgaccagct tggctgggag aatgaccact atttcctgag gaaaagtagg ctgtcctctg   1620 

ggctggtgac ggcggctctc tatgggcgct tgtatgagag ggactaggaa aagtgtgtgg   1680 

tctgtggggc ggactctggc catcatgttg acagccttgc acttccacag gctacaagcc   1740 

ctgggaccat ctttctgcct taagggcagg agcccacttc tgtggcacca gattccaccc   1800 

tgggaactgt ggaaaaagta ggagaggcag attccctcag tgtcttcctc ttaaacctca   1860 

atcatcgaga cattaggggg tattttctgt tcacatttat ctttttcaag ccacatcttc   1920 

ctctaacaaa tctctcagta tgcgattggt ctcaagctaa aacaaaaata aatgtcagtt   1980 

tatattttac acatccaaaa aaaaaaaaaa aaaaaa                             2016 

 
           
             2  
             1000  
             DNA  
             Homo sapiens  
           
            2 

cttccctccc tcccgggcgc taaaaggaaa accccccgac ccccatcgcc catttctact     60 

cgtctccaag acaacatcgc ggtccccgcc agcttccgta ggagcctttc attccaggaa    120 

ggtccatcgt acttgcgttt tcagggcctg agcgatgact ttagcaccag acacctgctc    180 

ctcgctgcgc tctgcgaagt tactgtccgg catcttccac cgaaaagctc taagcactca    240 

cgcagccggc aaacaagcgg aatcatccag caaggcaaac gcgaagtcgg cagcacgcca    300 

cctctggtac tgcacctctg acggacagga gggcaaccaa ctgccttaaa caacgggaag    360 

gaagaggcgg tctaaattcg tccacttccc gggagaggtg agaatgtaaa cacgcgcatt    420 

ctccaatcag aactgcgctc tcttctcggc tcctccattc gcgcgccaga atgccagagg    480 

gaggcgggac tagcaggaga ttgctgccta tgcaaagcag gtaagaagcc gaactctgag    540 

gcctctcgcc attgtctccg agtcggccag ctggagcgtt ttcggggctg taaagggaga    600 

atggcgcatg cgcatattca gggcggaagg cgcgctaaga gcaggtacgg agggggcgtg    660 

gtgcggcgcg gagggggtgt ggtaagggcg tgcggtccag accccgcccc gggcgcccag    720 

ttggacttgg ggagggctgc gcaaaggctg ccgggagctg ggaagcccgg cgcgcgtcgt    780 

ttgctggggc tgtttgtgcg ttgctgctgt gctaccgcgt tgcgttttct aggcatttac    840 

ttacacgctt tgtggtttac gctctcataa ccttgtggtt ttatagtcct taaattattg    900 

tagcgcacgt tacttaaatc cagaagcaga tgtgtacccc agcaagagat aaaatgacgc    960 

tcagagtcag tagatccaga ccgtgcctga gatcctgaat                         1000 

 
           
             3  
             12990  
             DNA  
             Homo sapiens  
           
            3 

tctcactggc tgctcttatg atccagaatg gaagaggatg aggacaaatg cagggggatg     60 

ttaggagacc actaagcagg tccctgtcat ttctctctct gtgattcctt ttgctgccac    120 

tttctccttg tccccttggc tccagccccc tgctgtcctt gctgtttctt gcacccgccc    180 

gtgaggcatg ctcctgcctc aggctctctg cctgccgtgc tctcacctcg cagacccacg    240 

tgatttcctc ccgaaccccc ttcaggcctc ggtccaaaca tcaccctctc atcgaggtct    300 

tccttgacca cactgcttaa aattgtcccc ctcgcctcac cttaccacct tcactgcctc    360 

atttctcctt tgtgcttaat caccatctca cacagttatc cagagcacga gccccacgag    420 

gaagggtctg tcttgtccac tgttggaccc ctgacaccta acccctgggg ctggcccaca    480 

gttgggaatt gcatggctgt ctgaagccag caccttcctg gttctgctgt tccctggaag    540 

tggggttcag tacaccccac gagccaaggc cctcatctca gagggcgtgc acatggttgc    600 

ctcaattgtg ctttcacact ggacccttcc tgcagtttac tctcctatgt cagatgccct    660 

tcaatgaaag caagtacatt gccaccttgt cacacctcta gttaccattt tctttatggt    720 

ccaggtcctg accagctcta aagatgggtc agctgctgta tttccaagaa ccccatgact    780 

tccagggccc cttgttccca ggaaggcccc gcaccccagt cgtcccgtgc ataacctcac    840 

gggccagcac ctggtagctg ctggaaggct tctgggggat gcctgggccc ctccagccct    900 

acctctagtc ctgccacttt acaactgagc acctcccgct gctgcctgct gaacccttca    960 

gagtccttgc cacaggccct atttactttt tcctgagaag gtatgtgtga tgccaaagag   1020 

agaaaagcag catttgctaa tttgggtaaa atgtttcttt gggaagggca aattgatgta   1080 

cagttgtccc tcagtatctg ttgggactgg ttccaggacc ccgtggatac cgaaaccctc   1140 

agatgctcaa gtcccttata tcaaatggtg tagaatttac atgtaaccta aacacttcct   1200 

cctgtacact ttaaatcatc tctagatccc ttacactacc taatacaaac taaatgctat   1260 

ataaataatt gttatactgt attttttaaa ttgctattat tttttattgt tgtgctatta   1320 

tttttattgg gctccctcct ccccctccca ccccagtaat ttcaatccaa ggttggtgga   1380 

atccatggat gcagaaacaa tggatactga gggccggctg actagaccta tttttattgt   1440 

aattactcat tctcttctac ctcattaagc ttggttcttt caagcatgaa tacctggttg   1500 

aatctgcata acctcttatt atcgacctac ctttgttcac acacaaatga ttgccactta   1560 

gagctctcct accgggctcc ttttgtaaaa taaattttat cttctccaga tagaaagaaa   1620 

tgtgatacct tgcggatttt gcggtttcct tgttttgctg gattgcaagt cctttagaca   1680 

taaacaaatg ttctgtaccc cgacatcggt ggtacccagc tctcagttga gaaagagatg   1740 

taatgtgaat gccactcttg gccccaggca ctcctcactt gcccccactg gggactggtt   1800 

cataccctcc tctcctggcc tcagtttcca tacctcttag tgaggccttt gcgttatact   1860 

tcagaaatat tgtcagtatg actttgaaga tgaaagtttg cccccaaaat cactctctgt   1920 

tatcattgtg aaaccagaga tggaatggaa aaatgggttt ctgagacatt ttaaattatt   1980 

ccttgcttgt ctttagaggc aaaattcaga taagaaagct tatcaattat acttttgttt   2040 

ctactcaaaa actcatgact gctcactcaa agactccttg ttcttatctt aaaacattgt   2100 

ttacagtgtc ccagatgaat ggcaacaaat cctggggttt ggtgtttgga tggtacattt   2160 

ccctggggaa agaaataaca gtttgagatg gaacggggtt ggggtgggag aatacttctc   2220 

attctgagga atatttaatt ttgccaagat gagcatttct agttcttagt ctgttgacga   2280 

aaagagctat ggtttgtttc tggaactttt gataaaaaat aaagaaattt gtagcctggg   2340 

gagtttggtt ttaaaatgca aacacaggag ttatgagttg agacttggac agggtgtcat   2400 

tttcttttta aagggcagca atatgattct ttgatttgtc tttgttatct tgacttttaa   2460 

tccggattcc tgggcagttg ttcagcccca ggacatctcc atgggcaggt ggcctggcct   2520 

tggcacacta cccagtaaat ctctgcctga gaggacgctt tagctgggag gccaggctga   2580 

tttttaaagg cagaattgga ctatttactc taaaacagta atgcacactg tttagaaaga   2640 

aacattccta ttctgggagg aaggaggaga cacacagaag tatcatttat ttctagtctt   2700 

ttctggtaga agctatgaag ctgagtttac tctctggaaa tttgtagttt attttctaga   2760 

aaattgcatt ttatcactgc aaaaaggatt ttatttccaa atgagtaggc ttttgagcaa   2820 

gagttttgga gtcacagaga tggggttaag aaagtgataa tgtgcaatgg cgattctcaa   2880 

gttcaaggag aaaaaataac atgcttttat tgggatactt tgcttgtcta taaaagaaag   2940 

tagctattgg catttatgta gaagtcagca gtttcttggc accaaataaa taattttgtg   3000 

ctgaataaag ggagagttat ccatagtatt tattactaac caaagaaatg cagggagaat   3060 

tgtaattcat taggttttga tggccaggaa agccaagctg tgttattagg gtcatgacaa   3120 

tcacagacat tacggatggc tgacctgtag tatggataga gggcagaggg tagagtgtga   3180 

aatatatcac agaattatgt caaataatct ggatagttac tactgcttaa aatctaagtg   3240 

cacagctaga aaagtgggta gtgacgcact acagtcttgc tgaacactgg gtaagaaaat   3300 

catagcaaac gttgagtctg ttttggaaat gttctaaaac cagactatta acacagtgag   3360 

ccattttaaa tgtggcttgc tacgtgtttg gagagaaaca catactcttt tattaggaac   3420 

atgaaacaaa ctctttgagc cgcagtatca ctgcgagtga gtttaattgc tgggattaat   3480 

aaatcacagc tgcaaacgtt aaattcttgg caggattctt tattcagctg ttttcccctt   3540 

gccccattac attccagatt tgtggtctgc attatgtctg gagccagaag taagcttgct   3600 

cttttcctct gcggctgtta cgtggttgcc ctgggagccc acaccgggga ggagagcgtg   3660 

gctgaccatc acgaggctga atattatgtg gctgccgtgt atgagcatcc atccatcctg   3720 

agtctgaacc ctctggctct catcagccgc caagaggcct tggagctcat gaaccagaac   3780 

cttgacatct atgaacagca agtgatgact gcagcccaaa aggcaagaat gctcctcgga   3840 

acctgagttt ctctcataca gagcagattg ctctttaccc cttgatcagt ggttgggtaa   3900 

tcccaggctt cctaccaccc tctgaaaaag catccaggta gttaacctga gttgagttag   3960 

tcagttgaat taggagcctt acccctcaga gagtggtccg tggaccggca tcccctggga   4020 

gcttgttaga aatacaaaat cttgggcggc accccagacc tactgaatca gaatgtgcat   4080 

tgcagcagga tccccaggtg atgctttcac atggcaagta tgagaagccc aggactagat   4140 

ccccagttct caagtgtggt tgtacataag aatcacgagg taagtggtaa acactatggc   4200 

tgcccgggtc ctggagagtc cgttgtaatt ggtgtggaag gggtgtggac tggcactggg   4260 

attgttttaa ggctccccag tgcagtctaa tgtgcagaaa aaatttgaaa gatgactggg   4320 

cgtgatgacc tctctgagtc attcgaagct tcactgaagt agtaagcatc tgcaagaatg   4380 

ccgtttgctc ccttcagact gtttgaggct cgtttccggt ctctatgtcg gactacgatc   4440 

agtctgagac cttcgcccag atagaactga ccccaaactg acaaagggaa ggtcagtgcc   4500 

agcctttgtg aaggcttcct ggttggcctg aatttcctgc tcccttcagg aagggtgggg   4560 

acaaaggaga ggcccccctg ggggcaaaga gggaaatatc agaggttgcc taagaaaatg   4620 

ccctgctgga aaacacaaac ccgaagggaa gtttgggctg taactctggt ggcagggtga   4680 

ccaagcgcag ctgcttgagg aagccctgct gtgcctcaac aggatgtaaa ctcattgtga   4740 

gcaacacttt cctgctctct gtgaacttaa agggcagaac cagcaggtcc tgccccaaac   4800 

agtccctgcc ttagagcagg gtggtcggga tggcctggac agccacagca attaaaaaat   4860 

tgcaacattt taaaatttta gtctataata tatatacaaa ggctatgtgt atggggtggg   4920 

ggggtgtttg ggggcagggg gtgtgtatgt gtgtataaca tgatgttgaa agggaacttg   4980 

aagacttggt ccagcttctt ttttttcaac caagaccaac ttttgcaagg gtgacacttt   5040 

tctttagtcc caacctgaca tacggtttct ccttgaacac cttcagtggc tcagactcac   5100 

aggtccgttt gttccaatgg tgggaacttc tgaacaggtc ttcctttcaa tgagcagcag   5160 

tcagcctccc cgtaactgcc accacgattc tatcgtcaga gctaaaggga gcaggaccgt   5220 

gtcccttatc acggcatgcc atcttctcca cctttgagga cagctgtcat gatccccctg   5280 

gcatctgtcc cccaggctgt atcctcagtc ccttccacag ttccttagga gactcagttt   5340 

ccaaaccttc tactgaagac ttccatgttt tctctgtgct cagaactgta tgcagctatc   5400 

ccgattctgt ctaataaggg cagggtagag aactctcacc tgtcgcattc tagatgttgt   5460 

ccccagaaaa ctgctggcag ccacatgtct cattatgggt gtataaggca cttgctgtca   5520 

actaaaacac cttttcacat gagcagacac acatgctgcc attgccatcc tgtacttata   5580 

aattataaag gtgattgatt taagctgagg gcaagacttc acatttatgc tgttaaattt   5640 

catcattcca gcctgttggg ctatttaggg atctttactg acatcccaag tatcagttac   5700 

ctttacgtca ttcacacata tgatacacac ctcatttatg tctatgctga agtcagtgta   5760 

aaaaaacccc aggctgtgcc ctcagacctc ctgatgacac tgatctccta gagggcaggc   5820 

attctcttga tagagatgtt tgcctgcatg gcactgagtc cagcacctga aatgtcatct   5880 

gcctcttgct tccctcccct atccaccgga ccattctgag acatttggca aatgacacac   5940 

tgaaacccag actgtggctg tagaattctc ctgcattcac ctttcaataa tctgccccca   6000 

gaggaaacac ttaacacggt tttgttgaaa ccacgccagc tgcacagcat cactccgtct   6060 

ctatttgttt tccaggggcc aggattaagc tgttgatatg atcactttta gaatttacag   6120 

atatctcagc tcccatacgt ggttatatgt tttttatttg tttgttttcc agcagcactt   6180 

ttattttcct tacacgatga catgttgctg gggcctattg ttctcacata acagtagaaa   6240 

accaaaattt gttgtcatct cttcaaagaa tcgagaattg catacagaaa aaccttacat   6300 

aaattaaaag gatgaataca tttacaggtg taaatgcaaa ccactttcaa ctcagacaag   6360 

taacagccca tggtgttctg gcagaaaaca tcagctaaga aaggaaactg ggtcctaagt   6420 

cttggacttt ccaaccctta cagaccggca gaacagaaac aactggttca ggagcccttg   6480 

ccagcctcca gagaaatccc agaacacgca gccctgacgt attaataccc tgcacagatc   6540 

agagactgct ggccacgcag actcaccaag ccacagactt gtcttccaca agcactttct   6600 

tatcttagcc acaaagtgac caagccacat gtactaaggg ttgaaatcaa agatatgtac   6660 

agggtattaa gcaaatctgg ttatatgttt taaaacaact tctaagacaa attgatggca   6720 

agtttgtgtg aaagttttat atcaaagttg ttataagagg ttcctgagca aaccaattga   6780 

aatacagtca tgcattgctt aatgacaggg atatgttctg aaaggatgca tcattaggcc   6840 

attgtgtcat tgtgcatgca tcatagcatg tacttacaca aacctacatg gtacggccta   6900 

ctatgcgcct aggctatatg gtatggccca ttgttcctag gctataaacc tttacagcat   6960 

attactgtac tgaacactgt aggcagttgt aacaagtggt aagcatttgt atatgtaaac   7020 

atagaaaagg tacaataaaa attcagtatt ataatcttat gggaccacca tcacatatgt   7080 

ggtctgtcat tgaccaaaat gtcatcatgc agtgcatgac tatatttctg tctcagtagg   7140 

ggcattcata ggggaaaaac ggagtctagt ttcaagatga ttaggctggg cagtcacttg   7200 

ggattgtaac cttcattcct cagaaggaag gggttcttga tctcattgag atctaccaga   7260 

aaattgctga agccatttat caagaatgca acttacttcc tagataggat tactcatcac   7320 

atcagaccca aaattttgcc cagctcaggt ttggttcctc tcctcattcc tggttgataa   7380 

taatctagta tgtatacata atttaaatgt tattctccat gaaaaaccaa agttttgttt   7440 

ttaataaaga aaaatgtcta tccaaatata attttcaaaa atctgaaaag atgactcata   7500 

caaatataga atgaataaag cttttattta attcattaat taaggaacca gtaagatggt   7560 

aaagctggtt caaaggaaaa ttcaaggaat ggaaatgtgt atatcagtca gtccagtgat   7620 

tgttgaaatg aatttcctaa tagatgcaaa actgggtaat gtcctatagg gcaaaacatt   7680 

gtaatctttg aggtgatctt ttaaatagca aagtcaaacg gtggtacatt ctccagctaa   7740 

ttaaagaata attgagtgag cctattaaac agtaccctag tataatttgg aaaggctgca   7800 

tctccatctt gccttatttt taggtttgag ataatttttc tttacatggt cattgctaag   7860 

tgtgcaatga gatgatactg tactggaagg aacatacatt ggtatagtat ttctggaaag   7920 

cagtttggca gtgtgtgtta agaacttaaa agtttaattt ttaggccagg tgctgtggct   7980 

catgcctgta atcccagcat tttgggggtc caaagcgggc ggatcacttg aggtcaggag   8040 

tttgagacca gcctgatggt gaaaccccat ctccactaaa aatacaaaat ttagccaggt   8100 

gtggtggcgc atgtctgtaa tcccagctac tcaggaggct gaggcacgag aattacttga   8160 

acccaggagg cggagattgc agtgagccga gatcacaaca ctgcactcca gcctgggcga   8220 

cagaccaaga ctctctctca aaaaacaaaa caaaaattaa aactctaatt tttataccct   8280 

ttgatccagt aatttcactt gtaagacttt attccaaaga aataatcaaa agatgcaatc   8340 

aaagatttgt gtgaagtgta taattatgca ataagtgttt tgagcacact atgcagatgg   8400 

tcaccacagt tttcttttta ttacaaaaag ttgggaacac ttcaaattcc aataatagag   8460 

gataaattat ggcgtcctct taaatatgat gtggccccat tacaaatgga tttttgaaag   8520 

tttttttttt ttcctttttt ttttgtggtg gagtttcact ttgtcaccca ggctggagtg   8580 

caatggtgcg atctcagctc accgcaacct ctgcctcccg ggttccagtg attctccagc   8640 

ctcagcctcc tgagtagctg ggattgcagg tgcccgctac catgcctggc taatttttgt   8700 

atttttagta gagacggggt ttcatcatgt tgggcaggct ggtcttgaac tcctgagctc   8760 

aggtgatctg cccacctggg cctcctgaag tgctgggatt acaggcgtga actgccatgc   8820 

ttggccgtat tttttaaagt tcttaatgag ggaagtcaag atgtaaaacc atatatttat   8880 

tattatctcc attatataca cacatacatg tatacagaga gaaaaagtaa tgaaaataac   8940 

caaaatatta acaataagta tctgtgttat agaattatga ttgttttttc ccgttttcca   9000 

aattttctac agtaaaactt ttgaagcttt tataaccagg aaaaaaattt aaaagtttgc   9060 

aatgcattcc agaaataagt gtctcaaact ttgctaattt gaattgttca tgccttctct   9120 

gcctgccttc tccaccttcc tccctggggc tggtgttccc ggcttgacat tttaaaccct   9180 

gtaagtggag agcagtggaa gaatgatgcc ccagccctga gagctgaggg cggccctgtt   9240 

tgtattttct taggttgctg tagatgtcac agggagttcc gggccatcac agccagggaa   9300 

cacaggatgt tgccaggtgt gggaaaaggc ctttagggtg gtcagagtcc cgaagggagc   9360 

ctcctaattc ccagttgggg aatggagatt tcaagcgagt tcttgtttcc aggctgagat   9420 

gagcacactt gcctcttacc cactggccca gtggatccta accttggcta caaatgagaa   9480 

tcacccgggg gacctttaaa caaacactgt tgccactatc ccacccacag tcaatcaaat   9540 

cagactttgt aggggtggtc ccggcatcag tggtttttca gaagttcctc aactgattta   9600 

aatgcacaat ggaagttgac aaccaccaga ctgaagatac cacgtgtgtt aatgggccca   9660 

atgtattcaa ggcccagtag ttggccccat ctcccctggt atcctaagaa ctctaaatcc   9720 

tttctagcta ttcgcttgtc aaactcctga gcttactttc aatggagctt acacattccc   9780 

tccttccctc acatgacccc aggcacagtt aatggttgtt cctagaggac tttgtctttg   9840 

ttccttgggg atcaggtgga gtgagacagt atccccaaga ctaagatctc tgaggagagt   9900 

aaagacacca tctctgtgcc tctggttcct gctacagagt aacttcctga tggttgccaa   9960 

aagaatgaac agaagaatga atgaatgcag cggttcttcc tgccatctga taacagacta  10020 

ttctttgatg ttttcatttt caggatgtac agattatagt gtttccagaa gatggcattc  10080 

atggattcaa ctttacaaga acatccattt atccattttt ggacttcatg ccgtctcccc  10140 

aggtggtcag gtggaaccca tgcctggagc ctcaccgctt caatgacaca gaggtgattc  10200 

ctgccttttt cctcagtagg ctgagggtac acagaggtga tctaagtcag ggaccagaag  10260 

ctgtgacatg ttaactaaga ttgataggag accttaacat ccccaaaatc caacccaaac  10320 

tcccaaagat ccatgtgcca catgttcatt ccattaaaga atgtctgacg ttacaaggca  10380 

gttattcatc tatggatctt tccatttatt aattacacaa taaatacagg aatgtatact  10440 

taaaccaaac caaaagtaaa aaaagaaaag ttcatcttca ccacagcctg cacctcatcc  10500 

catgcccttg cttagagaaa ctgccatcaa caatttgatg tgcattcagt tgtattcttt  10560 

tctatgcatt tcatagttat tgacatcctc tttttttttt tttttttgag atggagtctt  10620 

actctgccac ccaggctgga gcgcagtggc gcgatctcgg ctcactgcaa gctccgcctt  10680 

ctgggttcac gccattctcc tgcctcagcc tcccgagtag ctgggactac aggcatccac  10740 

caccacgccc ggctaatttt ttgtattttt agttgagatg gggtttcacc gtgttagcca  10800 

gggtggtctc aatctcctga cctcatgagc cacccgcctc agcctcccac agtgctggga  10860 

ttacaggcaa aaacctcatt tatttacacc tttttttcct ctaggtgctc cagcgcctga  10920 

gttgtatggc catcagggga gatatgttct tggtggccaa tcttgggaca aaggagcctt  10980 

gtcatagcag tgacccaagg tgcccaaaag atgggagata ccagttcaac acaaatgtcg  11040 

tgttcagcaa taatggaacc cttgttgacc gctaccgtaa acacaacctc tactttgagg  11100 

cagcattcga tgttcctctt aaagtggatc tcatcacctt tgataccccc tttgctggca  11160 

ggtttggcat cttcacatgc tttgatatat tgttctttga ccctgccatc agagtcctca  11220 

gagactacaa ggtgaagcat gttgtgtacc caactgcctg gatgaaccag ctcccactct  11280 

tggcagcaat tgagattcag aaagcttttg ctgttgcctt tggcatcaac gttctggcag  11340 

ctaatgtcca ccacccagtt ctggggatga caggaagtgg catacacacc cctctggagt  11400 

ccttttggta ccatgacatg gaaaatccca aaagtcacct tataattgcc caggtggcca  11460 

aaaatccagt gggtctcatt ggtgcagaga atgcaacagg tgaaacggac ccatcccata  11520 

gtaagttttt aaaaattttg tcaggcgatc cgtactgtga gaaggatgct caggaagtcc  11580 

actgtgatga ggccaccaag tggaacgtga atgctcctcc cacatttcac tctgagatga  11640 

tgtatgacaa tttcaccctg gtccctgtct ggggaaagga aggctatctc cacgtctgtt  11700 

ccaatggcct ctgctgttat ttactttacg agaggcccac cttatccaaa gagctgtatg  11760 

ccctgggggt ctttgatggg cttcacacag tacatggcac ttactacatc caagtgtgtg  11820 

ccctggtcag gtgtgggggt cttggcttcg acacctgtgg acaggaaatc acagaggcca  11880 

cggggatatt tgagtttcac ctgtggggca acttcagtac ttcctatatc tttcctttgt  11940 

ttctgacctc agggatgacc ctagaagtcc ctgaccagct tggctgggag aatgaccact  12000 

atttcctgag gaaaagtagg ctgtcctctg ggctggtgac ggcggctctc tatgggcgct  12060 

tgtatgagag ggactaggaa aagtgtgtgg tctgtggggc ggactctggc catcatgttg  12120 

acagccttgc acttccacag gctacaagcc ctgggaccat ctttctgcct taagggcagg  12180 

agcccacttc tgtggcacca gattccaccc tgggaactgt ggaaaaagta ggagaggcag  12240 

attccctcag tgtcttcctc ttaaacctca atcatcgaga cattaggggg tattttctgt  12300 

tcacatttat ctttttcaag ccacatcttc ctctaacaaa tctctcagta tgcgattggt  12360 

ctcaagctaa aacaaaaata aatgtcagtt tatattttac acatccacaa agcagtggct  12420 

tggggttttt tttttttttt ttatcttgtt gatcaagtga cacccaggac atgtaaatat  12480 

ttcataagcc ttaaacattt cctgaggtaa gaaacaagct ctcaaagcaa aagctcaatt  12540 

agaaatggcc cttgtgggga accttcccat tctggtcgac cagaactcta gccagatgaa  12600 

atggcaatgc tagcgccacc agcaacgtca gaaacgtaga ccttaaagcg gttttaaaaa  12660 

tagaaaagaa gcgttcctca catctgccag taatggaatt ttctgtcagt aaatggaatg  12720 

tgtaggcagg acctggaata actggagaga gtgcaacgct tcggggtgaa gggcgggtgg  12780 

ggactggaaa tgttgagacg ggggcagcca tgggaaggta tgagtaatag aattctttct  12840 

gtacgacaca gctcatccag ggattccagg ggaccttaat aaatcacggt agctttgggc  12900 

aagagttggg cacgtcgccc gactgtgcag gatggattga tgctggtatt aatttggtct  12960 

ggagccctat agaggatctc gttgctttga                                   12990 

 
           
             4  
             22  
             DNA  
             Homo sapiens  
           
            4 

gccccattac attccagatt tg                                              22 

 
           
             5  
             20  
             DNA  
             Homo sapiens  
           
            5 

gcccacctta tccaaagagc                                                 20 

 
           
             6  
             20  
             DNA  
             Homo sapiens  
           
            6 

gcttggctgg gagaatgacc                                                 20 

 
           
             7  
             20  
             DNA  
             Homo sapiens  
           
            7 

ggggaaagga aggctatctc                                                 20 

 
           
             8  
             20  
             DNA  
             Homo sapiens  
           
            8 

ctccagcgcc tgagttgtat                                                 20 

 
           
             9  
             20  
             DNA  
             Homo sapiens  
           
            9 

ctcatacacg gcagccacat                                                 20 

 
           
             10  
             19  
             DNA  
             Homo sapiens  
           
            10 

ggtgtcgaag ccaagaccc                                                  19 

 
           
             11  
             20  
             DNA  
             Homo sapiens  
           
            11 

cttgtagcct gtggaagtgc                                                 20 

 
           
             12  
             20  
             DNA  
             Homo sapiens  
           
            12 

acaggtgtcg aagccaagac                                                 20 

 
           
             13  
             21  
             DNA  
             Homo sapiens  
           
            13 

tccattattg ctgaacacga c                                               21 

 
           
             14  
             30  
             DNA  
             Homo sapiens  
           
            14 

tggtctgcat tatgtctgga gccagaagta                                      30 

 
           
             15  
             30  
             DNA  
             Homo sapiens  
           
            15 

tttgatgggc ttcacacagt acatggcact                                      30 

 
           
             16  
             29  
             DNA  
             Homo sapiens  
           
            16 

agggactagg aaaagtgtgt ggtctgtgg                                       29 

 
           
             17  
             30  
             DNA  
             Homo sapiens  
           
            17 

agggcataca gctctttgga taaggtgggc                                      30 

 
           
             18  
             30  
             DNA  
             Homo sapiens  
           
            18 

aggagccttg tcatagcagt gacccaaggt                                      30 

 
           
             19  
             20  
             DNA  
             Homo sapiens  
           
            19 

gcttgctctt ttcctctgcg                                                 20 

 
           
             20  
             21  
             DNA  
             Homo sapiens  
           
            20 

actacatcca cgtgtgtgcc c                                               21 

 
           
             21  
             20  
             DNA  
             Homo sapiens  
           
            21 

ctctatgggc gcttgtatga                                                 20 

 
           
             22  
             20  
             DNA  
             Homo sapiens  
           
            22 

tgaagcccat caaagacccc                                                 20 

 
           
             23  
             20  
             DNA  
             Homo sapiens  
           
            23 

tggtgaccaa tcttgggaca                                                 20