Multiplex amplification system and kit for X chromosome STR locus
1. A multiplex amplification system for X-chromosome STR loci selected from DXS8378, DXS7423, DXS10148, DXS10159, DXS10134, DXS7424, DXS9902, DXS10162, DXS7132, DXS10079, DXS6789, GATA165B12, DXS101, DXS10075, DXS10103, DXS10101, HPRTB, DXS6809, GATA31E08, DATA172D05, DXS6803, DXS10074, s10135, DXS6810, Amelogenin, DXS6795, DXS6800, DXS7133, DXS10164, DXS6807, DXS9895, DXS 9877, and 1 Amelogenin sex recognition loci, comprising 33 primer pairs for specific multiplex amplification of the X-chromosome STR loci.
2. The multiplex amplification system of claim 1, wherein the nucleotide sequences of 33 pairs of the primer pairs are represented by SEQ ID number 1 to SEQ ID number 66, respectively.
3. The multiplex amplification system according to claim 2, wherein the final concentration of each primer is 0.06. mu.M, 0.08. mu.M, 0.11. mu.M, 0.08. mu.M, 0.10. mu.M, 0.13. mu.M, 0.07. mu.M, 0.11. mu.M, 0.34. mu.M, 0.12. mu.M, 0.06. mu.M, 0.29. mu.M, 0.10. mu.M, 0.16. mu.M, 0.21. mu.M, 0.06. mu.20. mu.M, 0.06. mu.M, 0.M, 0.06. mu.M, 0.29. mu.M, 0.10. mu.10. mu.M, 0.16. mu.21. mu.M, 0.02. mu.M, 0.20, 0.19. mu.M, 0.11. mu.11. mu.M, 0.11. mu.11. mu.M, 0.11. mu., 0.40. mu.M, 0.45. mu.M, 0.05. mu.M, 1.50. mu.M, 0.50. mu.M, 0.47. mu.M, 0.16. mu.M, 0.49. mu.M, 0.10. mu.M, 0.31. mu.M.
4. The multiplex amplification system of any one of claims 1 to 3, wherein at least one primer of the primer pair corresponding to each locus is labeled with a fluorescent dye at its 5' end.
5. The multiplex amplification system of claim 4, wherein the primer pairs for the specific multiplex amplification DXS8378, DXS7423, DXS10148, DXS10159 and DXS10134 loci are in a first group, the primer pairs for the specific multiplex amplification DXS7424, DXS9902, DXS10162, DXS7132, DXS10079 and DXS6789 loci are in a second group, the primer pairs for the specific multiplex amplification GATA165B12, DXS101, DXS10075, DXS10103, DXS10101, hpb and DXS6809 loci are in a third group, the primer pairs for the specific multiplex amplification GATA31E08, DXS 172D05, DXS6803, DXS10074, DXS10135 and DXS6810 loci are in a fourth group, the primer pairs for the specific multiplex amplification Amelogenin, DXS6795, DXS6800, DXS7133, DXS 6864, DXS 9807, DATA 9895, DXS 9877 and DXS981 loci are in the same group, and the fluorescent marker sets are different from each other.
6. The multiplex amplification system of claim 5, wherein said fluorescent dye label is selected from the group consisting of 6-FAM, HEX, TAMRA, ROX, VIC, PET, NED, TAZ and SIZ.
7. The multiplex amplification system of claim 6, wherein the first set of fluorescent dyes is labeled 6-FAM, the second set of fluorescent dyes is labeled HEX, the third set of fluorescent dyes is labeled SUM, the fourth set of fluorescent dyes is labeled LYN, and the fifth set of fluorescent dyes is labeled PUR.
8. The multiplex amplification system of claim 1, comprising 10.0. mu.L of reaction mixture, 1.0. mu.L of hot start Taq enzyme, 5.0. mu.L of a mixture of 33 pairs of the primer pairs, and an appropriate amount of sdH2O; the reaction mixed solution comprises Tris-HCl and MgCl2KCl, dNTP and BSA; the final concentration of Tris-HCl in the reaction was 50mM, MgCl2The final reaction concentration of (1) was 2.0mM, the final reaction concentration of KCl was 50mM, the final reaction concentration of dNTP was 0.2mM, the final reaction concentration of BSA was 0.8mg/mL, and the content of hot start Taq enzyme was 5U/. mu.L.
9. A kit comprising a multiplex amplification system according to any one of claims 1 to 8, together with an allelic typing standard and an internal fluorescent molecular weight standard.
10. Use of the multiplex amplification system according to any one of claims 1 to 8 or the kit according to claim 9 for forensic individual identification, suspect pedigree screening, prenatal diagnosis and/or paternity testing.
Background
Short Tandem Repeat (STR) is a DNA sequence with length polymorphism existing in human genome, the core sequence of the STR is generally composed of 2-6 bases, and the core sequence presents length polymorphism due to different numbers of Tandem repeats among different individuals. On average, one STR locus exists in every 6-10kb of human genome DNA, and the high-degree polymorphic repeated sequences widely distributed in the human genome become effective genetic markers for individual identification and paternity test. STR fragments are small and easy to amplify, and are suitable for detecting trace and degrading biological detection materials, and amplification conditions of all loci are similar, so that composite amplification and automatic detection can be realized, and the STR fragments have the advantages of sensitivity, accuracy, rapidness, large information quantity and the like, and are very suitable for establishing a DNA database. Currently, methods used in forensic work include autosomal detection and/or Y chromosome detection. However, in the daily case examination process, when the relationship between the mother and the child is identified, and the relationship between the sister with the missing parents, the relationship between the sister and the father and the alien mother is identified, and the relationship between the grandmother and the grandmother is identified, the identification is difficult to be completed quickly at low cost only by the existing detection technology.
Disclosure of Invention
The present invention provides a multiplex amplification system and kit for an X chromosome STR locus, which solves one or more technical problems in the prior art and provides at least one useful choice or creation condition.
A composite amplification system for X chromosome STR gene locus comprises reaction mixed liquid, hot start Taq enzyme, primer mixture and sdH2O, and the extracted DNA to be detected. Wherein the primer pair comprises a 33 pair primer pair that specifically multiplex amplification of 32X chromosome STR loci selected from DXS8378, DXS7423, DXS10148, DXS10159, DXS10134, DXS7424, DXS9902, DXS10162, DXS7132, DXS10079, DXS6789, GATA165B12, DXS101, DXS10075, DXS10103, DXS10101, HPRTB, DXS6809, GATA31E08, DATA172D05, DXS6803, DXS10074, DXS10135, DXS6810, Amelogenin, DXS6795, DXS6800, DXS7133, DXS 101s 10164, DXS6807, DXS9895, DXS 9877, and DXS981, and 1 Amelogenin sex recognition loci.
When the diad is detected, the X-STR is more visual and effective than the autosomal STR in the identification of the relationship between a mother and a child as well as between a father and a mother. Especially in the identification of the genetic relationship of father and daughter, the X chromosome STR genetic marker can be well applied. The male contains one X chromosome, and the X-STR exists in the form of a haplotype that is completely derived from the mother and is completely inherited by the daughter. In the genetic relationship between female and female, the X chromosome is the same as the normal staining, and does not play more roles. However, in special cases, for example, where two individuals are not mothers or females but autosomes cannot be excluded, the addition of X-chromosome genetic markers has a very good effect on exclusion. Also, according to the unique genetic mode of the X chromosome, the X chromosome STR plays an important role in relation identification of sisters with deletion of parents, sisters with same parents and grandparents-grandchildren. The average exclusion rate of the loci of the X chromosome STR is superior to that of the autosomes due to the single loci, so that the X chromosome STR has a certain effect in the detection of putrefaction degradation test materials, and the loci of the front small segments can provide certain information.
The composite amplification system of the X chromosome STR loci can realize simultaneous amplification and detection of 33 loci in a single tube, so that the X chromosome STR loci can be used for realizing individual identification and quick investigation of suspects by X chromosome locus information, can also be used for prenatal diagnosis and/or paternity identification, and can provide effective technical support for quick detection and field detection of forensic DNA. Specifically, the information for the selected loci is shown in table 1:
TABLE 1 information on each locus
In some embodiments, the final concentration of the specific amplification primers is between 0.025 μm and 0.51 μm. Specific primer sequences and concentrations are shown in table 2:
TABLE 2 primer sequences and corresponding concentrations
In some embodiments, at least one primer of the primer pair for each locus has a fluorescent dye label at its 5' end. And further grouping the 32X chromosome STR loci and 1 Amelogenin sex recognition locus, wherein each group has the same fluorescent dye mark, and the fluorescent dye marks of the groups are different from each other, so that the detection efficiency can be improved.
In some embodiments, to avoid the overlapping of the peak positions of the products among different loci, it is ensured that the typing results of 32 loci in the re-amplification system do not affect each other, and therefore, the loci are reasonably arranged and grouped according to the length of the amplified fragments of the loci; the primer pairs for specific multiplex amplification of the DXS8378, DXS7423, DXS10148, DXS10159 and DXS10134 loci were assigned to the first group, the primer pairs for specific multiplex amplification of the DXS7424, DXS9902, DXS10162, DXS7132, DXS10079 and DXS6789 loci were assigned to the second group, the primer pairs for specific multiplex amplification of the GATA165B12, DXS101, DXS10075, DXS10103, DXS10101, HPRTB and DXS6809 loci were assigned to the third group, the primer pairs for specific multiplex amplification of the GATA31E08, DXS 172D05, DXS6803, DXS10074, DXS10135 and DXS680 loci were assigned to the fourth group, and the primer pairs for specific multiplex amplification of the Amelogenin, DXS6795, DXS6800, S7133, DXS10164, DXS6807, DXS9895, DXS 988377 and DXS 9877 loci were assigned to the fifth group.
Optional fluorescent dye labels include 6-FAM, HEX, TAMRA, ROX, VIC, PET, NED, TAZ, SIZ. In some embodiments, the first set of fluorescent dyes is labeled 6-FAM, the second set of fluorescent dyes is labeled HEX, the third set of fluorescent dyes is labeled SUM, the fourth set of fluorescent dyes is labeled LYN, and the fifth set of fluorescent dyes is labeled PUR. In addition, the molecular weight internal standard selects orange fluorescence SIZ as the fluorescent dye label, i.e., the sixth group of fluorescent dyes.
In some embodiments, the multiplex amplification system for the X chromosome STR locus totals 25.0. mu.L, including 10.0. mu.L of reaction mixture, 1.0. mu.L of hot start Taq enzyme, 5.0. mu.L of a mixture of 33 pairs of the primer pairs, 0.125-5.0 ng of genomic DNA, and the balance sdH2And (4) complementing O. Further, the reaction mixture comprises Tris-HCl and MgCl2KCl, dNTP and BSA.
Specifically, the final reaction concentration of Tris-HCl is 50mM, MgCl2The final reaction concentration of (1) is 2.0mM, the final reaction concentration of KCl is 50mM, the final reaction concentration of dNTP is 0.2mM, the final reaction concentration of BSA is 0.8mg/mL, and the hot start Taq enzymeThe content of (b) is 5U/. mu.L.
The amplification program of the composite amplification system of the X chromosome STR locus comprises the following steps: pre-denaturation at 95 deg.C for 5 min; 30 cycles of 94 ℃ for 10 s, 59 ℃ for 1 min and 68 ℃ for 1 min; final extension, 20 min at 60 ℃; keeping the temperature at 4 ℃. The fragment length of the resulting amplification product was about 500 bp.
The invention also provides a kit, which comprises the composite amplification system of the X chromosome STR locus, a carrier for extracting human genome DNA, an allele typing standard substance and a fluorescent molecular weight internal standard substance. The carrier contains human genome DNA extracted by any one of a Chelex method, a magnetic bead extraction method or an organic extraction method, and/or human blood or oral cells collected by any one of a filter paper, an FTA card, a cotton swab and gauze.
The multiplex amplification system and the kit of the X chromosome STR locus provided by the invention can be applied to forensic individual identification, suspect family investigation, prenatal diagnosis, an X chromosome database and/or paternity test. Can simultaneously amplify 33 gene loci in a single tube, and is far beyond the upper limit of other composite amplification systems or kits of the same type. The detection fragment of the STR kit is promoted to 500bp by combining with the analysis of AGCU Marker SIZ-500, and the DNA sample which can be suitable for the detection fragment is from seminal plaque, salivary plaque, tissue, hair follicle, blood stain or blood, and the like, so that the requirements of public security practice on the rapid detection and on-site detection capability of the DNA of a forensic doctor can be met, and effective technical support can be provided.
Drawings
FIG. 1 is a schematic diagram of the arrangement of genetic marker loci of the kit;
FIG. 2 is a typing chart of a standard DNA 9948 of the kit shown in example 4;
FIG. 3 is a typing chart of the standard DNA 9947A of the kit according to example 4;
FIG. 4 is a diagram of Allelic Allelic Ladder typing according to the example 4;
FIG. 5 is a typing map of the sister sample described in example 5;
FIG. 6 is a typing map of a sister sample according to example 5;
FIG. 7 is a typing chart of sample 1 described in example 6;
FIG. 8 is a typing chart of sample 2 described in example 6.
The following detailed description of the embodiments of the present invention is provided in connection with the examples to facilitate understanding and understanding of the technical solutions.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 screening of X chromosome STR loci
Selecting STR loci with high discriminative power in X chromosome, and performing calculation on the allele frequency of each of DXS10011, DXS10074, DXS10075, DXS10077, DXS10079, DXS101, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10148, DXS10159, DXS10161, DXS10162, DXS10163, DXS10164, DXS10165, DXS6789, DXS6795, DXS6799, DXS6800, DXS6801, DXS6804, DXS6807, DXS6809, DXS6810, DXS7132, DXS7133, DXS7423, DXS7424, DXS 77, DXS8378, DXS981, DXS9902, DXS 2500, GATA165B12, GATA D05, GATA31E 52, and DXS 8332, and performing calculation on the allele frequency of each of STR loci (allele frequency) of each of the STR loci, and the allele frequency of each of the STR loci are calculated according to the allele frequency of each of the identified genes of the selected STR loci, and the genes: DXS8378, DXS7423, DXS10148, DXS10159, DXS10134, DXS7424, DXS9902, DXS10162, DXS7132, DXS10079, DXS6789, GATA165B12, DXS101, DXS10075, DXS10103, DXS10101, HPRTB, DXS6809, GATA31E08, DATA172D05, DXS6803, DXS10074, DXS10135, DXS 0, DXS6795, DXS6800, DXS7133, DXS10164, DXS6807, DXS9895, DXS8377, DXS981 indicate that these 32X chromosome STR loci have high genetic polymorphisms and good allele frequency distribution in the human population (as shown in table 1).
Example 2 design of specific primers and selection of primer concentrations
With the increase of the number of primers in the multiplex amplification system, the mutual interference between primers of different loci is more and more serious, and the dynamics of a reaction system becomes more and more complex, so that a large number of primer sequences need to be designed to carry out complex tests and to search for the specific concentration ratio between the primers in the multiplex amplification system, and finally more human X chromosome STR loci are compounded under the condition of not reducing the specificity and sensitivity of the kit.
Specifically, the method comprises the following steps:
(1) specific amplification primer design
Downloading a locus sequence by using a UCSC or NCBI website through a locus name or an X chromosome position; next, primer design was performed based on the sequences flanking each locus repeat unit. Before designing a primer, the property of a target sequence to be detected must be analyzed, and a region with high conservation and uniform base distribution is selected for primer design; when designing the primers, the length of the primers is ensured to be moderate, the primers have similar physical characteristics and reaction kinetic characteristics, Tm values and GC contents are similar, and dimers cannot be formed among the primers; meanwhile, the applicability of the primer to different test materials in the composite amplification is also considered, and the requirement of the amplification balance of different test materials is ensured. The Tm values of the upstream and downstream primers are consistent and do not exceed 2 ℃. Avoid the continuous over 3 base complementary pairing between the 3' ends of any two primers. Avoid the primer sequence from binding to the non-specific region of the template sequence. The primer sequence needs to be reduced in the matching region in addition to the specific primer binding region.
(2) Primer validation
Firstly, performing a primer single amplification experiment, and excluding primers with non-specificity and low amplification efficiency; secondly, carrying out a single-color small multiplex amplification experiment, namely, separately mixing and testing each group of primers, and as with single amplification, removing primers which generate non-specificity and have low amplification efficiency; finally, large multiplex amplification experiments are carried out, namely all five groups of primers are mixed and tested. In a single-color small multiplex amplification experiment, the condition that a primer with good single amplification experiment effect cannot be matched with other primers in the same group occurs. In this case, it is impossible to predict whether the sequence of a single primer having a poor effect should be adjusted or the sequences of the remaining primers in the same group should be adjusted. Similarly, the situation that the primer group with good single amplification effect and other primer groups influence each other also occurs in large-scale double-amplification experiments. In addition, the detection efficiency can be changed by adjusting the concentration of the primer. The inventors have surprisingly found that the above problems can be solved by using different concentrations of the same set of upstream and downstream primers, respectively, and 33 pairs of primer sequences and corresponding concentrations are finally obtained as shown in Table 2. The final kit genetic marker locus layout is shown in figure 1.
Example 3 determination of amplification System
Besides the specific amplification primer of the gene locus, the reaction system of the kit also comprises reaction mixed liquor, hot start Taq enzyme and sdH2O, the reaction mixture comprises Tris-HCl and MgCl2KCl, dNTP and BSA, and the concentration of each component can be adjusted according to needs.
In particular, Mg can be amplified in a PCR amplification system2+And designing an orthogonal experiment for optimizing the concentration and the dNTPs concentration to prepare a PCR reaction mixed solution, and adding the PCR reaction mixed solution into a PCR system. The final PCR system compositions are shown in Table 3.
TABLE 3 PCR reaction amplification System
Wherein the final reaction concentration of each component in the reaction mixture is 50mM Tris-HCl, 50mM KCl and 2.0mM MgCl20.2mM dNTPs, 0.8mg/mL BSA; primers for 33 loci and their primer concentrations are shown in table 3. Furthermore, 33 loci were grouped and fluorescently labeled as follows: DXS8378, DXS7423, DXS10148, DXS10159 and DXS10134 are the first group, the fluorescent dye is marked as 6-FAM; DXS7424, DXS9902, DXS10162, DXS7132, DXS10079 and DXS6789 are in the second group, and the fluorescent dye is HEX; GATA165B12, DXS101, DXS10075, DXS10103, DXS10101, HPRTB and DXS6809 as a third group, fluorescenceThe photo-dye label is SUM; GATA31E08, DATA172D05, DXS6803, DXS10074, DXS10135 and DXS6810 are in the fourth group, and the fluorescent dye is labeled LYN; amelogenin, DXS6795, DXS6800, DXS7133, DXS10164, DXS6807, DXS9895, DXS8377 and DXS981 are a fifth group, and the fluorescent dye is PUR.
Example 4 methods of use of the kits of the present application
Methods of use of the kits of the present application may include a PCR amplification reaction step, wherein the PCR amplification reaction may include an amplification reaction program. In some embodiments, the amplification reaction procedure differs, as do the amplification products. Specifically, when the 33 pairs of primers are amplified in a single-tube PCR system, the difference of amplification efficiency is easily larger due to different amplified fragments and different amplification marker primers, and further, the difference of amplification peak heights is obvious during detection, so that different amplification procedures need to be tested to ensure that different detection materials can obtain correct DNA typing in a wider temperature range in order to improve the difference of amplification peak heights, improve the problem of amplification balance among different loci, meet the adaptability of different detection materials and the tolerance of different annealing temperatures.
The following is for illustrative purposes only and does not limit the intended scope of the present application. First, the cycle number test: 28 cycles, 29 cycles, 30 cycles and 31 cycles are respectively tested, and experiments prove that the optimal cycle is 30; and secondly, annealing temperature test: respectively testing 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃ and 62 ℃, and the optimal temperature is 59 ℃ through experimental verification; thirdly, the problem of large difference of amplification efficiency of large and small fragments is solved by testing the extension time and the extension temperature in the cycle, so that the amplification performance of the kit is improved better, and the final amplification procedure is as follows in table 4:
TABLE 4 amplification procedure
Based on the above-obtained preferred amplification procedure, the amplification steps of the kit of the present application are as follows:
1) placing a PCR amplification tube containing a PCR amplification system on a thermal cycler;
2) the procedure as described in table 4 was selected for amplification;
3) storing the amplified product in dark;
4) after electrophoresis is finished, centrifuging the PCR amplification product at 3000rpm for 5 min, mixing 1 mu L of centrifuged PCR amplification product or kit allele typing standard with 0.5 mu L of fluorescent molecular weight internal standard AGCU Marker SIZ-500 and 12 mu L of deionized formamide to avoid generating bubbles, carrying out denaturation at 95 ℃ for 3 min, carrying out ice bath for 3 min, carrying out electrophoresis detection by a genetic analyzer, and analyzing electrophoresis data by using fragment analysis software GeneMapper. Wherein, the typing results of the DNA standard control 9948, the DNA standard control 9947A and the allele typing standard are respectively shown in figure 2, figure 3 and figure 4.
Example 5 example analysis 1
A pair of sister samples were selected and subjected to holomorphic analysis using the kit of the present application, with the results shown in fig. 5, fig. 6 and table 5:
TABLE 5 sister sample genotyping
According to "identification of biological holomorphic relationship implementation Specification SF/Z JD 0105002-2014", the status consistency scoring method of X chromosome STR loci is shown in Table 6:
TABLE 6 calculation of State consistency scores for STR loci of the X chromosome
After genotyping comparison and status consistency scoring of the X chromosome STR locus, according to the genotyping of table 5 and the calculation method of table 6, the result is that the IBS value is 55, which belongs to the IBS interval of 32 or more, i.e., the two samples detected in this example tend to be identified as being human siblings, which is consistent with the fact.
Example 6, example analysis 2
Two unrelated individual samples were taken and subjected to a whole sibling analysis using the kit of the present application, with the results shown in fig. 7, fig. 8 and table 7:
TABLE 7 genotyping of unrelated individual samples
After genotyping comparison and status consistency scoring of the X chromosome STR locus, according to the genotyping of table 7 and the calculation method of table 6, the result is that the IBS value is 13, which belongs to the IBS ≤ 21 interval, i.e., the two samples detected in this example tend to be identified as human unrelated individuals, consistent with the fact.
The above examples prove that the detection results are consistent with the facts, and the multiplex amplification system and the kit for the X chromosome STR loci provided by the technical scheme can be used in actual combat.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Sequence listing
<110> Guangdong Huamei Zhongyuan Biotech Co., Ltd; wuxi Zhongde Mei Biotechnology Ltd; guangzhou city criminal science and technology institute
<120> composite amplification system and kit for X chromosome STR locus
<130> 2021
<160> 66
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial sequence
<400> 1
gacagttcac ccctccttag gca 23
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<400> 2
aagagcgaaa ctccaactca aa 22
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence
<400> 3
ctctttctat aagtcttcc 19
<210> 4
<211> 19
<212> DNA
<213> Artificial sequence
<400> 4
gcctggcaca tagtaggtg 19
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence
<400> 5
acagagggag attctgtctc aa 22
<210> 6
<211> 17
<212> DNA
<213> Artificial sequence
<400> 6
gcttccttta atctttt 17
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence
<400> 7
tgcagtgagc caagattgtg c 21
<210> 8
<211> 26
<212> DNA
<213> Artificial sequence
<400> 8
gaaacacact tgatcatggt ggatta 26
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<400> 9
cagctactcc ggaggctgag 20
<210> 10
<211> 24
<212> DNA
<213> Artificial sequence
<400> 10
catgctgcat ttttttaatt tatt 24
<210> 11
<211> 19
<212> DNA
<213> Artificial sequence
<400> 11
aaagttatgc cagccactg 19
<210> 12
<211> 21
<212> DNA
<213> Artificial sequence
<400> 12
ctggtccagg aacacgcaca t 21
<210> 13
<211> 21
<212> DNA
<213> Artificial sequence
<400> 13
taattggaga gactgaggaa c 21
<210> 14
<211> 21
<212> DNA
<213> Artificial sequence
<400> 14
tctttgttat tataaaaagc a 21
<210> 15
<211> 22
<212> DNA
<213> Artificial sequence
<400> 15
cacacatcac caggcagttt cc 22
<210> 16
<211> 19
<212> DNA
<213> Artificial sequence
<400> 16
ttcttttgat taagcaggt 19
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence
<400> 17
gcctctggga cctgcatgag 20
<210> 18
<211> 19
<212> DNA
<213> Artificial sequence
<400> 18
tttacaaaac tcattctta 19
<210> 19
<211> 17
<212> DNA
<213> Artificial sequence
<400> 19
ctgtaatccc agctact 17
<210> 20
<211> 16
<212> DNA
<213> Artificial sequence
<400> 20
tgttgttgag ttcagt 16
<210> 21
<211> 18
<212> DNA
<213> Artificial sequence
<400> 21
ttgctcctca agcttgca 18
<210> 22
<211> 18
<212> DNA
<213> Artificial sequence
<400> 22
cactgtggtt gttggcaa 18
<210> 23
<211> 21
<212> DNA
<213> Artificial sequence
<400> 23
tttctaagcc aagtattgga c 21
<210> 24
<211> 21
<212> DNA
<213> Artificial sequence
<400> 24
acattcagaa ataatgttta a 21
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence
<400> 25
tcacatttta ctctaaatca 20
<210> 26
<211> 21
<212> DNA
<213> Artificial sequence
<400> 26
gcaaatcact ccatggcaca t 21
<210> 27
<211> 16
<212> DNA
<213> Artificial sequence
<400> 27
acaggttgtc attaga 16
<210> 28
<211> 19
<212> DNA
<213> Artificial sequence
<400> 28
tgcttgggcc tgtactggg 19
<210> 29
<211> 18
<212> DNA
<213> Artificial sequence
<400> 29
tggctctcag gcatttga 18
<210> 30
<211> 18
<212> DNA
<213> Artificial sequence
<400> 30
ccacatgcaa aagaatga 18
<210> 31
<211> 18
<212> DNA
<213> Artificial sequence
<400> 31
cagatatggc tgtgacaa 18
<210> 32
<211> 17
<212> DNA
<213> Artificial sequence
<400> 32
tgacaggggc cctgtac 17
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence
<400> 33
agctttcagc ttaaactttt 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence
<400> 34
acagataata cacatcccca 20
<210> 35
<211> 19
<212> DNA
<213> Artificial sequence
<400> 35
cctgaaaggc atgaaaaga 19
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence
<400> 36
gaaccttagt ttataatctt 20
<210> 37
<211> 22
<212> DNA
<213> Artificial sequence
<400> 37
aatcttgttt atcatactag at 22
<210> 38
<211> 24
<212> DNA
<213> Artificial sequence
<400> 38
tatcagctga cagagcacag agat 24
<210> 39
<211> 22
<212> DNA
<213> Artificial sequence
<400> 39
atacaattac tgtatgatcc ag 22
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence
<400> 40
ttattttggg aacataaaag 20
<210> 41
<211> 22
<212> DNA
<213> Artificial sequence
<400> 41
aggatatgaa tccattacat ga 22
<210> 42
<211> 21
<212> DNA
<213> Artificial sequence
<400> 42
agacacagta tgatgtagaa t 21
<210> 43
<211> 18
<212> DNA
<213> Artificial sequence
<400> 43
tttttgccat ttgttgtg 18
<210> 44
<211> 19
<212> DNA
<213> Artificial sequence
<400> 44
ggtctcagtg cccctcaga 19
<210> 45
<211> 17
<212> DNA
<213> Artificial sequence
<400> 45
aatagaggtt agtattt 17
<210> 46
<211> 19
<212> DNA
<213> Artificial sequence
<400> 46
acacctctct ggatagcat 19
<210> 47
<211> 21
<212> DNA
<213> Artificial sequence
<400> 47
tatggtccat aaaaatctgt t 21
<210> 48
<211> 22
<212> DNA
<213> Artificial sequence
<400> 48
gcctcggctt ggattacagg ca 22
<210> 49
<211> 17
<212> DNA
<213> Artificial sequence
<400> 49
tacgacccta ttccggt 17
<210> 50
<211> 19
<212> DNA
<213> Artificial sequence
<400> 50
agtctgtata cgtccctac 19
<210> 51
<211> 22
<212> DNA
<213> Artificial sequence
<400> 51
caacatcaaa acagaaatgc at 22
<210> 52
<211> 18
<212> DNA
<213> Artificial sequence
<400> 52
ggttttcagc tcctaggt 18
<210> 53
<211> 21
<212> DNA
<213> Artificial sequence
<400> 53
actctttctc tttatttctc a 21
<210> 54
<211> 17
<212> DNA
<213> Artificial sequence
<400> 54
tcagcttgca gagggcc 17
<210> 55
<211> 23
<212> DNA
<213> Artificial sequence
<400> 55
ctagtcagtt tgtaccttag taa 23
<210> 56
<211> 23
<212> DNA
<213> Artificial sequence
<400> 56
ctttattttt aacggtgttc atg 23
<210> 57
<211> 22
<212> DNA
<213> Artificial sequence
<400> 57
atatatttgt gttgactttg tc 22
<210> 58
<211> 19
<212> DNA
<213> Artificial sequence
<400> 58
aaaatcctaa acaaccaaa 19
<210> 59
<211> 19
<212> DNA
<213> Artificial sequence
<400> 59
aacttaaagg ccatggctc 19
<210> 60
<211> 19
<212> DNA
<213> Artificial sequence
<400> 60
aatttataaa ttagacttt 19
<210> 61
<211> 17
<212> DNA
<213> Artificial sequence
<400> 61
ctgtgatcca gagctcc 17
<210> 62
<211> 17
<212> DNA
<213> Artificial sequence
<400> 62
ctcccaccag gtccctc 17
<210> 63
<211> 19
<212> DNA
<213> Artificial sequence
<400> 63
gacccttctg caacagttc 19
<210> 64
<211> 18
<212> DNA
<213> Artificial sequence
<400> 64
tgctttgtat ttatagtc 18
<210> 65
<211> 21
<212> DNA
<213> Artificial sequence
<400> 65
aatcaattag agcagattca t 21
<210> 66
<211> 18
<212> DNA
<213> Artificial sequence
<400> 66
tgtcttgcta cacacctc 18
