CN114292923B - Genetic marker of 191 human micro-haplotype loci, amplification primer and application thereof - Google Patents
Genetic marker of 191 human micro-haplotype loci, amplification primer and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of forensic genetics identification, and particularly discloses a 191 human micro-haplotype locus genetic marker, an amplification primer and application thereof. The 191 human micro-haplotype locus genetic markers provided by the invention combine the advantages of STR and SNP, and have the advantages of short amplified fragment, low mutation rate, good polymorphism and no stutter peak. The genetic relationship identification of the 191 human micro-haplotype loci provided by the invention can independently solve the two-layer genetic relationship identification between two individuals, meets the requirement of only two ginseng and identification in the two-level genetic relationship such as grandpa grandis, nephew or nephew, and the like, has extremely high efficiency of an identification system, and has good repeatability, extremely high sensitivity and stability. In addition, the method has remarkable degradation resistance in forensic identification and extremely high application value in forensic practical examination cases.
Description
Technical Field
The invention relates to the technical field of forensic genetics identification, in particular to a 191 human micro-haplotype locus genetic marker, an amplification primer and application thereof.
Background
Short tandem repeats (short tandem repeat, STR) and single nucleotide polymorphisms (single nucleotide polymorphisms, SNP) are common genetic markers for forensics. STR becomes a mainstream genetic marker for individual identification and kindred relation identification due to the characteristics of high polymorphism and wide distribution, and plays an irreplaceable role in practical case inspection. Compared with STR, the SNP mutation rate is low, the amplified fragment is short, ancestral information can be provided, the phenotype characteristics of the individual can be predicted, and the method has more advantages in degradation detection material analysis and geographical ancestral inference. Although STR and SNP play important roles in judicial practice, they have great limitations and cannot fully meet the requirements of forensics. For example, the length part of the highly degraded material fragment is smaller than 100bp, and the fragment length of the existing kit STR is mostly in the range of 100 bp-450 bp; for ultra-trace samples with very small amounts of DNA template, stutter peaks often appear due to replication slippage during STR amplification, alleles cannot be distinguished from stutter using capillary electrophoresis (capillary electrophoresis, CE); when STR analysis is used for mixed materials, uncertainty is easily caused by the influence of the peak of the stutter and the mixing proportion of the materials. For SNPs, most SNPs are alleles, single SNPs have low polymorphism and insufficient information, so that the number of detection sites needs to be increased to achieve similar performance to STRs, however, recombination of excessive sites may affect the balance of amplification efficiency between loci, and thus allele loss occurs.
Genetic relationship identification is one of two major tasks of forensic evidence. The corresponding identification specifications (paternity identification technical specification GB/T37223-2018, biological isotactic relation identification technical specification SF/T0117-2021 and biological grandchild relation identification specification SF/Z JD 0105005-2015) of the traditional grandchild identification, isotactic cell identification and parent identification can be referred to for practical application, wherein grandchild identification has strict requirements on an identified person participating in identification, namely that grandparents, children and child parents are required to participate in identification at the same time, which is indispensable. Full sibling authentication is also a method that can only distinguish full siblings of the parent and unrelated individuals, whereas paternity authentication requires at least one party of parents of children to participate in authentication. The above identification content with standard reference can meet the requirements of daily commercialized kits. However, parents of children often encounter in actual examination cases to go out or cannot participate in authentication, and ancestor relationship (only grand two ginseng and authentication), half sibling authentication, tertile authentication, girl authentication and aunt outer 29989 are required to be authenticated, and the authentication belongs to two-level genetic relationship authentication and is one of the difficulties of the current forensic research.
Disclosure of Invention
Aiming at the problems of the conventional forensic genetic identification method, the invention provides 191 human micro-haplotype locus genetic markers, and amplification primers and application thereof, wherein the 191 human micro-haplotype locus genetic markers are used for forensic genetic identification, can meet the daily forensic examination case requirement, can independently solve the two-level genetic relationship identification between two individuals, and have extremely high accuracy, sensitivity and stability.
In order to achieve the above purpose, the embodiment of the invention adopts the following technical scheme:
a 191 human micro-haplotype locus genetic marker comprising the following 191 human micro-haplotype loci:
compared with the prior art, the 191 human micro-haplotype loci provided by the invention are genetically marked as a plurality of linked SNP, combine the advantages of STR and SNP, and have the advantages of short amplified fragment, low mutation rate, good polymorphism and no stutter peak. In practical application, the genetic markers of 191 human micro-haplotype loci provided by the invention can be used for identifying individuals, and can also be used for carrying out genetic relationship identification, including two-level genetic relationship identification between two individuals, so that the requirements of identification of only two ginseng such as grandmother, half sibling, nephew and the like are met, and the identification system has extremely high efficiency, good repeatability, extremely high sensitivity and stability. The 191 human micro-haplotype loci provided by the invention are used for forensic identification, have remarkable degradation resistance, and have extremely high application value in forensic practical examination.
The 191 human micro-haplotype loci provided by the invention accord with Hardy-Weinberg equilibrium in Hebei Han population, are in a linkage equilibrium state and mutually independent, and can independently solve the problem of two-level genetic relationship identification between two individuals.
The invention also provides primers for amplifying the 191 human micro-haplotype loci genetic markers, wherein the sequences of the primers are respectively as follows:
1)SEQ ID NO.1-2；2)SEQ ID NO.3-4；3)SEQ ID NO.5-6；4)SEQ ID NO.7-8；5)SEQ ID NO.9-10；6)SEQ ID NO.11-12；7)SEQ ID NO.13-14；8)SEQ ID NO.15-16；9)SEQ ID NO.17-18；10)SEQ ID NO.19-20；11)SEQ ID NO.21-22；12)SEQ ID NO.23-24；13)SEQ ID NO.25-26；14)SEQ ID NO.27-28；15)SEQ ID NO.29-30；16)SEQ ID NO.31-32；17)SEQ ID NO.33-34；18)SEQ ID NO.35-36；19)SEQ ID NO.37-38；20)SEQ ID NO.39-40；21)SEQ ID NO.41-42；22)SEQ ID NO.43-44；23)SEQ ID NO.45-46；24)SEQ ID NO.47-48；25)SEQ ID NO.49-50；26)SEQ ID NO.51-52；27)SEQ ID NO.53-54；28)SEQ ID NO.55-56；29)SEQ ID NO.57-58；30)SEQ ID NO.59-60；31)SEQ ID NO.61-62；32)SEQ ID NO.63-64；33)SEQ ID NO.65-66；34)SEQ ID NO.67-68；35)SEQ ID NO.69-70；36)SEQ ID NO.71-72；37)SEQ ID NO.73-74；38)SEQ ID NO.75-76；39)SEQ ID NO.77-78；40)SEQ ID NO.79-80；41)SEQ ID NO.81-82；42)SEQ ID NO.83-84；43)SEQ ID NO.85-86；44)SEQ ID NO.87-88；45)SEQ ID NO.89-90；46)SEQ ID NO.91-92；47)SEQ ID NO.93-94；48)SEQ ID NO.95-96；49)SEQ ID NO.97-98；50)SEQ ID NO.99-100；51)SEQ ID NO.101-102；52)SEQ ID NO.103-104；53)SEQ ID NO.105-106；54)SEQ ID NO.107-108；55)SEQ ID NO.109-110；56)SEQ ID NO.111-112；57)SEQ ID NO.113-114；58)SEQ ID NO.115-116；59)SEQ ID NO.11-118；60)SEQ ID NO.119-120；61)SEQ ID NO.121-122；62)SEQ ID NO.123-124；63)SEQ ID NO.125-126；64)SEQ ID NO.127-128；65)SEQ ID NO.129-130；66)SEQ ID NO.131-132；67)SEQ ID NO.133-134；68)SEQ ID NO.135-136；69)SEQ ID NO.137-138；70)SEQ ID NO.139-140；71)SEQ ID NO.141-142；72)SEQ ID NO.143-144；73)SEQ ID NO.145-146；74)SEQ ID NO.147-148；75)SEQ ID NO.149-150；76)SEQ ID NO.151-152；77)SEQ ID NO.153-154；78)SEQ ID NO.155-156；79)SEQ ID NO.157-158；80)SEQ ID NO.159-160；81)SEQ ID NO.161-162；82)SEQ ID NO.163-164；83)SEQ ID NO.165-166；84)SEQ ID NO.167-168；85)SEQ ID NO.169-170；86)SEQ ID NO.171-172；87)SEQ ID NO.173-174；88)SEQ ID NO.175-176；89)SEQ ID NO.177-178；90)SEQ ID NO.179-180；91)SEQ ID NO.181-182；92)SEQ ID NO.183-184；93)SEQ ID NO.185-186；94)SEQ ID NO.187-188；95)SEQ ID NO.189-190；96)SEQ ID NO.191-192；97)SEQ ID NO.193-194；98)SEQ ID NO.195-196；99)SEQ ID NO.197-198；100)SEQ ID NO.199-200；101)SEQ ID NO.201-202；102)SEQ ID NO.203-204；103)SEQ ID NO.205-206；104)SEQ ID NO.207-208；105)SEQ ID NO.209-210；106)SEQ ID NO.211-212；107)SEQ ID NO.213-214；108)SEQ ID NO.215-216；109)SEQ ID NO.217-218；110)SEQ ID NO.219-220；111)SEQ ID NO.221-222；112)SEQ ID NO.223-224；113)SEQ ID NO.225-226；114)SEQ ID NO.227-28；115)SEQ ID NO.229-230；116)SEQ ID NO.231-232；117)SEQ ID NO.233-234；118)SEQ ID NO.235-236；119)SEQ ID NO.237-238；120)SEQ ID NO.239-240；121)SEQ ID NO.241-242；122)SEQ ID NO.243-244；123)SEQ ID NO.245-246；124)SEQ ID NO.247-248；125)SEQ ID NO.249-250；126)SEQ ID NO.251-252；127)SEQ ID NO.253-254；128)SEQ ID NO.255-256；129)SEQ ID NO.257-258；130)SEQ ID NO.259-260；131)SEQ ID NO.261-262；132)SEQ ID NO.263-264；133)SEQ ID NO.265-266；134)SEQ ID NO.267-268；135)SEQ ID NO.269-270；136)SEQ ID NO.271-272；137)SEQ ID NO.273-274；138)SEQ ID NO.275-276；139)SEQ ID NO.277-278；140)SEQ ID NO.279-280；141)SEQ ID NO.281-282；142)SEQ ID NO.283-284；143)SEQ ID NO.285-286；144)SEQ ID NO.287-288；145)SEQ ID NO.289-290；146)SEQ ID NO.291-292；147)SEQ ID NO.293-294；148)SEQ ID NO.295-296；149)SEQ ID NO.297-298；150)SEQ ID NO.299-300；151)SEQ ID NO.301-302；152)SEQ ID NO.303-304；153)SEQ ID NO.305-306；154)SEQ ID NO.307-308；155)SEQ ID NO.309-310；156)SEQ ID NO.311-312；157)SEQ ID NO.313-314；158)SEQ ID NO.315-316；159)SEQ ID NO.317-318；160)SEQ ID NO.319-320；161)SEQ ID NO.321-322；162)SEQ ID NO.323-324；163)SEQ ID NO.325-326；164)SEQ ID NO.327-328；165)SEQ ID NO.329-330；166)SEQ ID NO.331-332；167)SEQ ID NO.333-334；168)SEQ ID NO.335-336；169)SEQ ID NO.337-338；170)SEQ ID NO.339-340；171)SEQ ID NO.341-342；172)SEQ ID NO.343-344；173)SEQ ID NO.345-346；174)SEQ ID NO.347-348；175)SEQ ID NO.349-350；176)SEQ ID NO.351-352；177)SEQ ID NO.353-354；178)SEQ ID NO.355-356；179)SEQ ID NO.357-358；180)SEQ ID NO.359-360；181)SEQ ID NO.61-362；182)SEQ ID NO.363-364；183)SEQ ID NO.365-366；184)SEQ ID NO.367-368；185)SEQ ID NO.369-370；186)SEQ ID NO.371-372；187)SEQ ID NO.373-374；188)SEQ ID NO.375-376；189)SEQ ID NO.377-378；190)SEQ ID NO.379-380；191)SEQ ID NO.381-382。
compared with the prior art, the primer for amplifying the 191 human micro-haplotype loci genetic markers can be specifically amplified in the same system to obtain the 191 micro-haploid loci, the primer pairs can not interfere with each other, the requirement of multiple PCR (polymerase chain reaction) can be met, the accuracy and the stability of forensic identification analysis by using the amplified loci as genetic markers are further improved, the operation steps of forensic identification are simplified, the difficulty of identifying two-level genetic relationship is particularly reduced, and the primer pair has extremely high application value.
The invention also provides application of the 191 human micro-haplotype locus genetic markers in forensic identification.
The invention also provides application of the 191 human micro-haplotype locus genetic markers in individual identification and identification or genetic relationship identification.
Preferably, the genetic relationship identification comprises paternity identification, isotactic cell identification and two-level genetic relationship identification in which only two individuals participate.
The invention also provides a forensic identification kit comprising primers for amplifying the genetic markers of the 191 human micro-haplotype loci.
Preferably, the forensic identification kit further comprises a buffer solution, a DNA polymerase and dntps.
The invention also provides a using method of the forensic identification kit, which comprises the following steps: extracting genome DNA of a sample to be detected as a template, carrying out PCR amplification by using the kit, carrying out a linker sequence PCR reaction on the obtained amplification product to obtain a multiple PCR library, and carrying out quantitative and second-generation sequencing detection analysis on the multiple PCR library to obtain a typing result of the micro-haplotype locus.
Preferably, the PCR amplification system comprises the following reagents and amounts:
the system for PCR reaction of the linker sequence comprises the following reagents and the dosage:
preferably, the genomic DNA concentration is 5ng-20ng.
Preferably, the concentration of each primer in the mixture of 191 human micro-haplotype locus amplification primers is 5-10. Mu.M.
Further preferably, the forensic identification kit may also incorporate primers corresponding to sex identification sites or other functional identification primers to meet the needs of the forensic identification kit for sex identification.
Drawings
FIG. 1 is a graph comparing the system performance of the forensic identification kit of example 4 of the test of example 6 of the present invention with that of the prior art kit in the same identification, wherein 2ND vs. UR: two-layer genetic relationship identification, 3RD vs. UR: three-layer genetic relationship identification; type of kit: MH: forensic identification kit in example 4, SIG: signature,42STR: 42STR kits constructed in the laboratory, COM A: SIG+42STR two kits are combined, COMB: the three kits are combined.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
Screening of micro-haplotype loci:
screening of 191 micro-haplotype loci on 22 autosomes, using crowd data of 103 beijing han groups and 105 southern han groups in the thousand-person genome database, screening according to the following specific conditions:
and (3) primary screening:
(1) Removing the recombination hot zone; (2) removing SNPs with heterozygosity less than 0.4; (3) The DNA fragment length is 250bp, 2-5 SNP, and 245479 micro-haplotype loci are screened out. And estimating haplotype and haplotype frequency of the micro-haplotype locus using phase.
And (3) re-screening:
the microsloid loci screened above were further screened:
(1) 3-5 SNP are arranged in 200bp of DNA fragment length; (2) an effective allelic factor greater than 2.5; (3) Looking at sequence features within +/-200 bp of the upstream and downstream of the cross micro-haplotype position on a UCSC browser, and eliminating micro-haplotype loci with long poly structures, repeat regions (repeat) and indels; (4) After the amplification primer is successfully designed, BLAT is carried out on the sequence on an Ensembl browser, and micro-haplotype locus sequences which can be aligned at a plurality of positions on a genome are removed; (5) To reduce the possibility of linkage, the physical distance of the microsloid loci located on the same chromosome is greater than 5Mb.
The total 191 micro-haplotype loci which are correctly typed can be obtained by the conditions and successfully sequenced, and the optimized 191 pairs of primers can meet the requirement of high-throughput amplification without generating interference.
Naming of micro-haplotype loci
At present, no standard naming mode exists for the micro-haplotype loci internationally, and the naming mode proposed by Kidd professor has certain limitation, so that the unification of the micro-haplotype loci among laboratories is not facilitated. The laboratory provides a novel micro-haplotype locus naming method, and the name of the micro-haplotype is observed to intuitively know the positions of the micro-haplotype locus on several chromosomes, including several SNP and the positions of the SNP on the chromosomes. The naming method proposed by us comprises micro-haplotype English letter abbreviation "mh", chromosome number and position of each SNP in a locus. For example, mh1-1987049/096/145/156, "mh" is a microsloid abbreviation to distinguish different genetic markers, "1" is the locus on chromosome 1, "1987049" is the position of the first SNP of a locus on the chromosome, "096/145/156" indicates the last digits of the second, third and fourth SNPs, respectively, on the chromosome that are not identical to the first SNP position. The naming method is visual and clear, is favorable for unification of micro-haplotype loci among laboratories, or has difference in polymorphism among different groups along with the increase of research groups, and can clearly know the increase or deletion of SNP in the micro-haplotype loci. Through the crowd and specific screening conditions, the 191 human micro-haplotype loci are finally obtained, and through detection and analysis, the advantages of STR and SNP are combined, and the method has the advantages of short amplified fragment, low mutation rate, good polymorphism and no stutter peak.
Example 2
The set of human micro-haplotype loci screened in example 1 comprises the following 191 human micro-haplotype loci:
example 3
The sequences of the 191 pairs of primers amplifying the 191 human micro-haplotype loci in example 2 are as follows:
1)SEQ ID NO.1-2；2)SEQ ID NO.3-4；3)SEQ ID NO.5-6；4)SEQ ID NO.7-8；5)SEQ ID NO.9-10；6)SEQ ID NO.11-12；7)SEQ ID NO.13-14；8)SEQ ID NO.15-16；9)SEQ ID NO.17-18；10)SEQ ID NO.19-20；11)SEQ ID NO.21-22；12)SEQ ID NO.23-24；13)SEQ ID NO.25-26；14)SEQ ID NO.27-28；15)SEQ ID NO.29-30；16)SEQ ID NO.31-32；17)SEQ ID NO.33-34；18)SEQ ID NO.35-36；19)SEQ ID NO.37-38；20)SEQ ID NO.39-40；21)SEQ ID NO.41-42；22)SEQ ID NO.43-44；23)SEQ ID NO.45-46；24)SEQ ID NO.47-48；25)SEQ ID NO.49-50；26)SEQ ID NO.51-52；27)SEQ ID NO.53-54；28)SEQ ID NO.55-56；29)SEQ ID NO.57-58；30)SEQ ID NO.59-60；31)SEQ ID NO.61-62；32)SEQ ID NO.63-64；33)SEQ ID NO.65-66；34)SEQ ID NO.67-68；35)SEQ ID NO.69-70；36)SEQ ID NO.71-72；37)SEQ ID NO.73-74；38)SEQ ID NO.75-76；39)SEQ ID NO.77-78；40)SEQ ID NO.79-80；41)SEQ ID NO.81-82；42)SEQ ID NO.83-84；43)SEQ ID NO.85-86；44)SEQ ID NO.87-88；45)SEQ ID NO.89-90；46)SEQ ID NO.91-92；47)SEQ ID NO.93-94；48)SEQ ID NO.95-96；49)SEQ ID NO.97-98；50)SEQ ID NO.99-100；51)SEQ ID NO.101-102；52)SEQ ID NO.103-104；53)SEQ ID NO.105-106；54)SEQ ID NO.107-108；55)SEQ ID NO.109-110；56)SEQ ID NO.111-112；57)SEQ ID NO.113-114；58)SEQ ID NO.115-116；59)SEQ ID NO.11-118；60)SEQ ID NO.119-120；61)SEQ ID NO.121-122；62)SEQ ID NO.123-124；63)SEQ ID NO.125-126；64)SEQ ID NO.127-128；65)SEQ ID NO.129-130；66)SEQ ID NO.131-132；67)SEQ ID NO.133-134；68)SEQ ID NO.135-136；69)SEQ ID NO.137-138；70)SEQ ID NO.139-140；71)SEQ ID NO.141-142；72)SEQ ID NO.143-144；73)SEQ ID NO.145-146；74)SEQ ID NO.147-148；75)SEQ ID NO.149-150；76)SEQ ID NO.151-152；77)SEQ ID NO.153-154；78)SEQ ID NO.155-156；79)SEQ ID NO.157-158；80)SEQ ID NO.159-160；81)SEQ ID NO.161-162；82)SEQ ID NO.163-164；83)SEQ ID NO.165-166；84)SEQ ID NO.167-168；85)SEQ ID NO.169-170；86)SEQ ID NO.171-172；87)SEQ ID NO.173-174；88)SEQ ID NO.175-176；89)SEQ ID NO.177-178；90)SEQ ID NO.179-180；91)SEQ ID NO.181-182；92)SEQ ID NO.183-184；93)SEQ ID NO.185-186；94)SEQ ID NO.187-188；95)SEQ ID NO.189-190；96)SEQ ID NO.191-192；97)SEQ ID NO.193-194；98)SEQ ID NO.195-196；99)SEQ ID NO.197-198；100)SEQ ID NO.199-200；101)SEQ ID NO.201-202；102)SEQ ID NO.203-204；103)SEQ ID NO.205-206；104)SEQ ID NO.207-208；105)SEQ ID NO.209-210；106)SEQ ID NO.211-212；107)SEQ ID NO.213-214；108)SEQ ID NO.215-216；109)SEQ ID NO.217-218；110)SEQ ID NO.219-220；111)SEQ ID NO.221-222；112)SEQ ID NO.223-224；113)SEQ ID NO.225-226；114)SEQ ID NO.227-28；115)SEQ ID NO.229-230；116)SEQ ID NO.231-232；117)SEQ ID NO.233-234；118)SEQ ID NO.235-236；119)SEQ ID NO.237-238；120)SEQ ID NO.239-240；121)SEQ ID NO.241-242；122)SEQ ID NO.243-244；123)SEQ ID NO.245-246；124)SEQ ID NO.247-248；125)SEQ ID NO.249-250；126)SEQ ID NO.251-252；127)SEQ ID NO.253-254；128)SEQ ID NO.255-256；129)SEQ ID NO.257-258；130)SEQ ID NO.259-260；131)SEQ ID NO.261-262；132)SEQ ID NO.263-264；133)SEQ ID NO.265-266；134)SEQ ID NO.267-268；135)SEQ ID NO.269-270；136)SEQ ID NO.271-272；137)SEQ ID NO.273-274；138)SEQ ID NO.275-276；139)SEQ ID NO.277-278；140)SEQ ID NO.279-280；141)SEQ ID NO.281-282；142)SEQ ID NO.283-284；143)SEQ ID NO.285-286；144)SEQ ID NO.287-288；145)SEQ ID NO.289-290；146)SEQ ID NO.291-292；147)SEQ ID NO.293-294；148)SEQ ID NO.295-296；149)SEQ ID NO.297-298；150)SEQ ID NO.299-300；151)SEQ ID NO.301-302；152)SEQ ID NO.303-304；153)SEQ ID NO.305-306；154)SEQ ID NO.307-308；155)SEQ ID NO.309-310；156)SEQ ID NO.311-312；157)SEQ ID NO.313-314；158)SEQ ID NO.315-316；159)SEQ ID NO.317-318；160)SEQ ID NO.319-320；161)SEQ ID NO.321-322；162)SEQ ID NO.323-324；163)SEQ ID NO.325-326；164)SEQ ID NO.327-328；165)SEQ ID NO.329-330；166)SEQ ID NO.331-332；167)SEQ ID NO.333-334；168)SEQ ID NO.335-336；169)SEQ ID NO.337-338；170)SEQ ID NO.339-340；171)SEQ ID NO.341-342；172)SEQ ID NO.343-344；173)SEQ ID NO.345-346；174)SEQ ID NO.347-348；175)SEQ ID NO.349-350；176)SEQ ID NO.351-352；177)SEQ ID NO.353-354；178)SEQ ID NO.355-356；179)SEQ ID NO.357-358；180)SEQ ID NO.359-360；181)SEQ ID NO.61-362；182)SEQ ID NO.363-364；183)SEQ ID NO.365-366；184)SEQ ID NO.367-368；185)SEQ ID NO.369-370；186)SEQ ID NO.371-372；187)SEQ ID NO.373-374；188)SEQ ID NO.375-376；189)SEQ ID NO.377-378；190)SEQ ID NO.379-380；191)SEQ ID NO.381-382。
example 4
A forensic identification kit comprising primers (191 pairs of primers in example 3) for amplifying the 191 human micro-haplotype loci described above, an IGT-EM808 polymerase mixture, an enhancement buffer NB, an enhancement buffer M, YF buffer B, IGT-I5 tag, and an IGT-I7 tag.
The application method of the forensic identification kit comprises the following steps:
extracting genome DNA in whole blood of a person to be detected, carrying out PCR amplification by taking the extracted genome DNA as a template, and carrying out a linker sequence PCR reaction on the obtained amplified product to obtain an identification product, namely a multiplex PCR library.
Wherein, the PCR amplification system comprises the following reagents and the dosage:
the amplification product obtained by the PCR amplification is subjected to first round magnetic bead purification, and the specific method is as follows:
1) Adding 27uL of AMPure XP magnetic beads subjected to room temperature equilibrium into a PCR tube (containing 30uL of PCR amplification product), and gently sucking and beating by a pipette for 20 times;
2) After 5min incubation at room temperature, the PCR tube is placed on a DynaMag-96 Side magnetic rack for 3min;
3) Removing the supernatant, taking the PCR tube off the magnetic rack, adding 50ul of YF bufferB into the tube, and gently sucking and beating for 20 times by using a pipettor;
4) After 5min incubation at room temperature, the PCR tube is placed on a DynaMag-96 Side magnetic rack for 3min;
5) Removing the supernatant, placing the PCR tube on a magnetic rack, adding 180ul of 80% ethanol solution into the tube, and standing for 30sec;
6) Removing the supernatant, placing the PCR tube on a magnetic rack, adding 180ul of 80% ethanol solution into the tube, standing for 30sec, and removing the supernatant;
7) Standing at room temperature for 3min to volatilize residual ethanol thoroughly;
8) Taking the PCR tube off the magnetic frame, adding 24ul of nucleic-free water, gently sucking and beating the re-suspended magnetic beads by a liquid shifter, avoiding generating bubbles, and standing for 2min at room temperature;
9) The PCR tube is placed on a magnetic rack again, and is kept stand for 3min;
10 13.5ul of supernatant was pipetted into a new 200ul PCR tube, the supernatant in the new PCR tube being the purified multiplex PCR amplification product.
The system for the PCR reaction of the linker sequence comprises the following reagents and the dosage:
the amplified product obtained by the PCR reaction of the linker sequence is subjected to a second round of magnetic bead purification, and the specific method is as follows:
1) Adding 27uL of AMPure XP magnetic beads which are balanced at room temperature into a PCR tube (an amplification product obtained by a PCR reaction with a connector sequence), and gently sucking and beating for 20 times by a pipette;
2) After 5min incubation at room temperature, the PCR tube is placed on a DynaMag-96 Side magnetic rack for 3min;
3) Removing the supernatant, taking the PCR tube off the magnetic rack, adding 50ul of YF bufferB into the tube, and gently sucking and beating for 20 times by using a pipettor;
4) After 5min incubation at room temperature, the PCR tube is placed on a DynaMag-96 Side magnetic rack for 3min;
5) Removing the supernatant, placing the PCR tube on a magnetic rack, adding 180ul of 80% ethanol solution into the tube, and standing for 30sec;
6) Removing the supernatant, placing the PCR tube on a magnetic rack, adding 180ul of 80% ethanol solution into the tube, standing for 30sec, and removing the supernatant;
7) Standing at room temperature for 3min to volatilize residual ethanol thoroughly;
8) Taking the PCR tube off the magnetic frame, adding 24uL of nucleic-free water, lightly sucking and beating the re-suspended magnetic beads by a liquid shifter, avoiding generating bubbles, and standing for 2min at room temperature;
9) The PCR tube is placed on a magnetic rack again, and is kept stand for 3min;
10 20uL of supernatant is sucked by a pipette and transferred into a new PCR tube, and the supernatant in the new PCR tube is the obtained multiplex PCR library.
Library quantification:
2ul of the prepared multiplex PCR library was used
3.0Fluorometer (Qubit dsDNA HS Assay Kit) for library concentration determination, library concentration was recorded. The concentration of normal library ranges from 5ng/uL to 50ng/uL, the library concentration being primarily related to the quality of the template.
Library quality detection:
the quantitative library was diluted to 5ng/uL and 10uL was used
GX Touch 24(DNA HiSens Chip&Reagent Kit) to detect the length of the library fragments, the distribution interval of the target fragments of the normal library is 280bp-420 bp.
The MiSeq FGx platform performs on-machine sequencing detection:
protocol a, reference MiSeq System Denature and Dilute Libraries Guide (Document #15039740 v10), denatures and dilutes the constructed library and PhiX, reference Illumina Experiment Manager Software Guide (Document #15031335v 08), edits sample forms using IEM software, reference MiSeq System Guide (Document #15027617 v05 Material#20000262) for on-machine sequencing. The sequencing reagent selected for this experiment was MiSeq Reagent Kit v or MiSeq FGx Reagent Kit (Micro Flow Cell & Standard Flow Cell), PE300 double ended sequencing.
Example 5
Repeatability test:
the kit of example 4 was repeatedly studied using cell line NA18530, one was to construct libraries three times each, while sequencing on the machine; the other is a library constructed once, and the library is sequenced on a machine three times respectively. Through data analysis and comparison, the typing of all 191 micro-haplotype loci of NA18530 is uniform, which shows that the micro-haplotype composite amplification kit (forensic identification kit) in the example 4 has good repeatability.
Sensitivity test:
the sensitivity study was performed on the kit of example 4 using cell line NA18530, with initial template amounts of 10ng, 5ng, 2.5ng, 1ng, 500pg, 250pg, 125pg, 62.5pg, respectively, repeated once for the first round of PCR amplification. Through data analysis, accurate typing of all micro-haplotype loci can still be obtained at a starting template amount of 250 pg. When the initial template amount was 125pg, there was allele loss at one micro-haplotype locus. Heterozygous equality of 11 and 13 microhaplotype loci in duplicate at a starting template amount of 62.5pg does not reach the set threshold, and there is allele loss of 3 microhaplotype loci in one sample at a starting template amount of 62.5 pg. In conclusion, the sensitivity of the micro-haplotype composite amplification kit in the embodiment 4 can reach 250pg, and the sensitivity is very good.
Stability test:
the stability study of the kit of example 4 was performed using the cell line NA18530, and included two types, one type of PCR inhibitor in which humic acid and hemoglobin were added to DNA, and the other type of degradation assay material was prepared using ultrasonic disruption.
(1) Humic acid PCR inhibitor, wherein the concentration of humic acid in 1ng/uL DNA solution is 25ng/uL, 50ng/uL, 75ng/uL and 100ng/uL respectively, and the initial template amount of the first round of PCR amplified DNA is 5ng and repeated once. Through data analysis, allele loss at both microsloid loci occurred at a humic acid concentration of 25 ng/uL.
(2) Hemoglobin PCR inhibitor, 1ng/uL DNA solution of hemoglobin concentration of 100uM, 200uM, 300uM and 400uM, the initial template amount of the first PCR amplified DNA was 5ng, repeated once. Through data analysis, allele loss at both microsloid loci occurred at a hemoglobin concentration of 200 uM.
(3) The degradation detection material is prepared by breaking DNA by ultrasound, breaking by ultrasound is carried out according to the conditions in table 1, and the library construction is carried out by quantitatively diluting Qubit to 5 ng/ul. GSTAR is adopted simultaneously for artificially prepared degradation detection materials TM The 25 kit (Beijing Ma Cheng and technology Co., ltd.) was used for composite STR amplification, and the ABI 3500 genetic analyzer was used for capillary electrophoresis analysis. Via GSTAR TM 25 kit amplified DNA, after 10min of ultrasonic disruption, serious locus imbalance, and after 20min of ultrasonic disruption, allele loss of the whole locus.
TABLE 1 conditions for ultrasonic disruption
However, the degraded DNA detected by using the micro-haplotype kit in example 4 can still be completely and accurately typed after 30min of ultrasonic disruption. The micro-haplotype locus amplification kit has good degradation resistance and can be applied to forensic practical examination cases.
Example 6
Population data was investigated on 221 Hebei han nationality healthy unrelated individuals using the kit of example 4, and 191 micro-haplotype loci were found to be in Hardy-Weinberg equilibrium in this population by Hardy-Weinberg equilibrium test.
In addition, locus independence analysis was performed to obtain 191 microsloid loci in linkage equilibrium, independent of each other.
The system efficacy of the kit of example 4, see in particular table 2, was calculated to confirm that the microsloid locus kit (forensic identification kit) could solve the same identification, paternity test and grandson identification problems.
TABLE 2 System efficacy of microsloid locus kits
| locus | TDP | CPEduo | CPEtrio | RGE |
| 191 pieces | 1-2.2025E-138 | 1-6.4440E-22 | 1-7.6383E-40 | 1-2.5273E-17 |
Note that: TDP: accumulating personal identification probabilities; CPEduo: bigeminal cumulative non-father exclusion probabilities;
CPEthio: triplet accumulating non-parent exclusion probabilities; RGE: average non-grandparent exclusion rate.
To test the application value of the kit in far-field relationship identification, the allele frequency of the crowd of 221 Hebei Han nationality unrelated individuals is utilized to simulate two-layer related individuals pairs, three-layer related pairs and 50 ten thousand pairs of unrelated individuals according to a genetic rule, two-layer and three-layer related identifications are respectively carried out, the Area (AUC) under a subject operation characteristic curve (ROC curve) is used as a system efficiency evaluation index, and the system efficiency evaluation index is matched with a commercialized kit ForenSeq TM DNA Signature Prep Kit(VeThe system efficacy of the two-generation sequencing kit consisting of the 42STR loci previously constructed in the laboratory in the same assay was compared, and the results are shown in fig. 1, in which 2nd vs. Two-layer genetic relationship identification, 3RD vs. UR: three-layer genetic relationship identification; type of kit: MH: forensic identification kit in example 4, SIG: signature,42STR: 42STR kits (published) constructed in this laboratory, COMA: SIG+42STR two kits are combined, COMB: the three kits are combined. Therefore, the system efficiency of the kit in the two-layer and three-layer genetic relationship identification is equivalent to that of the other two kits when the two kits are combined. In the kit in the embodiment 4, in the ancestor identification, the AUC reaches more than 0.9999, the problem of two-layer genetic relationship identification between two individuals can be independently solved, and the problem that the ancestor identification result cannot be realized because a parent sample cannot be provided is solved.
Example 7
A forensic identification kit comprising 191 pairs of primers in example 3 and 6 pairs of sex chromosome-located primers, the other reagents being the same as in the forensic identification kit of example 4.
The 6 primers located on sex chromosomes correspond to the 6 SNP sites for sex identification, and the 6 SNP sites and their corresponding 6 pairs of primers are shown in Table 3.
TABLE 3 6 SNP loci for sex identification and primers therefor
The forensic identification kit is used for high-throughput multiplex PCR detection (the detection method is the same as that of the embodiment 4), so that the accurate identification of two-level genetic relationships such as identity identification, paternity test, grandchild test, girl test or nephew test only involving two persons can be realized, and the accurate judgment of gender can be satisfied.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
SEQUENCE LISTING
<110> university of Hebei medical science
<120> 191 human micro-haplotype locus genetic markers, and amplification primer and application thereof
<130> 2021
<160> 394
<170> PatentIn version 3.5
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gccaacaatg ctttttctgc aag 23
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tcctccccac aggaaaggat aa 22
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agtggttctg gaagcttctc tg 22
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agaggctact ttccactccc tt 22
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taggaatgtg gaaagggcca ac 22
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actgttgaga gaagctgcct tc 22
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tgtgctagga gcatctacca ct 22
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ctgcatgaaa tgcaagggct ag 22
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agaccagaac tcagggttct ga 22
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catgcctccc acctgagtt 19
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tatgtaagga ttcgtgctgg gg 22
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cctccaggcc tagtatgaac cta 23
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cagtccagtg agagagacat gatg 24
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catcaacaaa aggtacgtgc act 23
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tgcactaaca accactggaa attg 24
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cagaacctac taacaatgca cttgag 26
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acgaaggaac acaaattgta ggc 23
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tggcgtttct tcatctctat tcact 25
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catcttttaa aagttctgac tcggtacag 29
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gtggcaaagc agttttcagc aa 22
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accttgtcag catgctgtta tatg 24
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cgttatgcaa tggctgagtt gaac 24
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caggtgcaga aaacacctac tct 23
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tgcagtgtta tttacaggtg ggag 24
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cctactctcc caattatgta tctctgt 27
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agctgtttgt ctttcaagtc tcct 24
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tctttaaggg ttcagtttgt tctcac 26
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cagtcccaca aaccaatcat cttt 24
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aacgggaatg taactaccaa agaaaac 27
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cagaagccct ttgtctcaag ca 22
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acctgagcac tgaagaacaa ttcag 25
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catggatgaa ataggagaat cccagta 27
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ctccggtatc tctgggtaag gt 22
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ctcaagagct ctgtccgtta cc 22
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tcctcacact gacataaaac aaaaagg 27
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ctgatcaagg catcagatct ctctt 25
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gggtttatcc actaaggaca cacaag 26
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catggagctt acagactgaa aagaaaat 28
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gtttcccaat tacacatatg cctacatc 28
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tgtggtagaa ttagagtcac atgtgag 27
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ggctgccaag ggaataataa agtc 24
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agtcaagctt aaatggattc tacatgt 27
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gggatctcct cattgtacct tgac 24
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gcttctcata ggggaaagga gtag 24
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cttaaaatag cacatctgat ttcccaga 28
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tgctttttca ggggcagaag ata 23
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tgttaaagcc acattcactt tttcctag 28
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gcttgagaat gtaataaccc cttctagt 28
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tttgtaatta tggctaaagt attagtgata gtc 33
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gacatgaata gtcattcacc ttaaattagg a 31
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cccgtggaaa gcatgatatg gta 23
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tgctttctgt gtgtgctgga t 21
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atgtgtgggg agactagtgc at 22
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agtgaatgca tgtgcacctc tc 22
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<211> 29
<212> DNA
<213> mh4-162585594/653/766-F
<400> 107
aggatgatca ttgcatttat tacatgtca 29
<210> 108
<211> 24
<212> DNA
<213> mh4-162585594/653/766-R
<400> 108
gtgtcatgac agttagctcc ctaa 24
<210> 109
<211> 26
<212> DNA
<213> mh4-174845656/790/795-F
<400> 109
ccttattacc cactcttcag gagcta 26
<210> 110
<211> 26
<212> DNA
<213> mh4-174845656/790/795-R
<400> 110
gcttattagc catgggattc ctatca 26
<210> 111
<211> 29
<212> DNA
<213> mh4-186640816/892/943-F
<400> 111
aaaatgtatt cttatgcttc accaaaacc 29
<210> 112
<211> 32
<212> DNA
<213> mh4-186640816/892/943-R
<400> 112
gttctgtgaa attagtaaag attatgggag aa 32
<210> 113
<211> 22
<212> DNA
<213> mh5-1860057/167/245/251-F
<400> 113
tctccacaga gactcttggt gg 22
<210> 114
<211> 15
<212> DNA
<213> mh5-1860057/167/245/251-R
<400> 114
<210> 115
<211> 27
<212> DNA
<213> mh5-12979828/868/954-F
<400> 115
gctcaatggt aacagcagaa atagttc 27
<210> 116
<211> 29
<212> DNA
<213> mh5-12979828/868/954-R
<400> 116
ctctcttcag atagactcct ttattggtg 29
<210> 117
<211> 27
<212> DNA
<213> mh5-29621406/425/521-F
<400> 117
cagagggatc aatataggca aatctca 27
<210> 118
<211> 25
<212> DNA
<213> mh5-29621406/425/521-R
<400> 118
cttccaggcc aattgttttt aacct 25
<210> 119
<211> 24
<212> DNA
<213> mh5-58793906/3912/4040-F
<400> 119
catctaggtg gaggaatcat tggt 24
<210> 120
<211> 24
<212> DNA
<213> mh5-58793906/3912/4040-R
<400> 120
cttgctgctg atttatgacc tctc 24
<210> 121
<211> 22
<212> DNA
<213> mh5-74544910/5024/5085-F
<400> 121
aacgatgtcc tcactgtcac ct 22
<210> 122
<211> 21
<212> DNA
<213> mh5-74544910/5024/5085-R
<400> 122
tgtcttcttg cctgcgatag c 21
<210> 123
<211> 21
<212> DNA
<213> mh5-85243205/264/270-F
<400> 123
tgttaacact gatggcgagc a 21
<210> 124
<211> 21
<212> DNA
<213> mh5-85243205/264/270-R
<400> 124
tgcattcgcc acaaattgct t 21
<210> 125
<211> 23
<212> DNA
<213> mh5-94697284/290/464-F
<400> 125
acgtggcaaa cataggttac tgt 23
<210> 126
<211> 24
<212> DNA
<213> mh5-94697284/290/464-R
<400> 126
tctcaacctt gtcttcagat gcaa 24
<210> 127
<211> 25
<212> DNA
<213> mh5-115023525/607/656-F
<400> 127
cagatgactt tttgtcacca acctt 25
<210> 128
<211> 22
<212> DNA
<213> mh5-115023525/607/656-R
<400> 128
aggtaggttc aaacgtggtg ag 22
<210> 129
<211> 22
<212> DNA
<213> mh5-125660025/080/185-F
<400> 129
accgtcacct ctttgtagga ct 22
<210> 130
<211> 23
<212> DNA
<213> mh5-125660025/080/185-R
<400> 130
agacctactg ggtccaggaa ata 23
<210> 131
<211> 23
<212> DNA
<213> mh5-142109741/816/894-F
<400> 131
tgtgccgggt gtattttaaa tgc 23
<210> 132
<211> 22
<212> DNA
<213> mh5-142109741/816/894-R
<400> 132
catcagttta ccacctggct ca 22
<210> 133
<211> 23
<212> DNA
<213> mh5-176568834/912/934-F
<400> 133
tctggactac gaggtaaaga gca 23
<210> 134
<211> 23
<212> DNA
<213> mh5-176568834/912/934-R
<400> 134
ctcacggaga tggtgacttt gaa 23
<210> 135
<211> 22
<212> DNA
<213> mh6-452728/732/759/778-F
<400> 135
tcccaggaga tgactgctac ag 22
<210> 136
<211> 22
<212> DNA
<213> mh6-452728/732/759/778-R
<400> 136
ctgtaggttc ttctccccag gt 22
<210> 137
<211> 24
<212> DNA
<213> mh6-27425930/6092/6105-F
<400> 137
tctcccagat ctcactctct tcag 24
<210> 138
<211> 24
<212> DNA
<213> mh6-27425930/6092/6105-R
<400> 138
aagccagatg acagaggtat gcta 24
<210> 139
<211> 22
<212> DNA
<213> mh6-36998194/260/349/350-F
<400> 139
tccaagccac tcatgtttct gt 22
<210> 140
<211> 24
<212> DNA
<213> mh6-36998194/260/349/350-R
<400> 140
agaggcaagg attgtgtttc tagg 24
<210> 141
<211> 23
<212> DNA
<213> mh6-45920098/185/285/296-F
<400> 141
gacaggtgct gcaaaatgtt ctc 23
<210> 142
<211> 23
<212> DNA
<213> mh6-45920098/185/285/296-R
<400> 142
gatgtttcat tgtcacacag ccc 23
<210> 143
<211> 25
<212> DNA
<213> mh6-55528157/158/284-F
<400> 143
ccaccaccaa ctctatcgat aatcc 25
<210> 144
<211> 24
<212> DNA
<213> mh6-55528157/158/284-R
<400> 144
atcgccttta agaggagtta ctgc 24
<210> 145
<211> 24
<212> DNA
<213> mh6-65542037/118/127-F
<400> 145
cctgggaacc tggaatgata gaag 24
<210> 146
<211> 25
<212> DNA
<213> mh6-65542037/118/127-R
<400> 146
gaagtgcctg gaaataatcc catag 25
<210> 147
<211> 21
<212> DNA
<213> mh6-77681361/439/445-F
<400> 147
ctgtttttca ccgccagggt a 21
<210> 148
<211> 21
<212> DNA
<213> mh6-77681361/439/445-R
<400> 148
catggcacaa ccagcaatca c 21
<210> 149
<211> 21
<212> DNA
<213> mh6-107704804/813/933-F
<400> 149
atggataggt ccccaggtca g 21
<210> 150
<211> 21
<212> DNA
<213> mh6-107704804/813/933-R
<400> 150
ccttttcgag aggctcctct g 21
<210> 151
<211> 23
<212> DNA
<213> mh6-144281002/030/122-F
<400> 151
gcgtagactg tgttttgttc agt 23
<210> 152
<211> 22
<212> DNA
<213> mh6-144281002/030/122-R
<400> 152
aggagctgaa atgtggaatc ca 22
<210> 153
<211> 28
<212> DNA
<213> mh6-152814204/243/272-F
<400> 153
gcagatatgg agaaaatctt cacattcc 28
<210> 154
<211> 28
<212> DNA
<213> mh6-152814204/243/272-R
<400> 154
gtaggttact gagctcagaa aatgaatg 28
<210> 155
<211> 21
<212> DNA
<213> mh6-165727439/571/575-F
<400> 155
ggagagagag gaggctttcc a 21
<210> 156
<211> 23
<212> DNA
<213> mh6-165727439/571/575-R
<400> 156
cttcctccta tctactccct gca 23
<210> 157
<211> 24
<212> DNA
<213> mh7-155353189/266/289-F
<400> 157
ctgggtctta ggaggaacta gttg 24
<210> 158
<211> 20
<212> DNA
<213> mh7-155353189/266/289-R
<400> 158
<210> 159
<211> 19
<212> DNA
<213> mh7-946204/291/360-F
<400> 159
tccaactcca cctccgtgt 19
<210> 160
<211> 22
<212> DNA
<213> mh7-946204/291/360-R
<400> 160
atcctcacaa tggaatggga cc 22
<210> 161
<211> 25
<212> DNA
<213> mh7-10789582/643/723-F
<400> 161
cttggctgta aacctctttt tggaa 25
<210> 162
<211> 27
<212> DNA
<213> mh7-10789582/643/723-R
<400> 162
tagccccaaa acactatttt tacatgc 27
<210> 163
<211> 26
<212> DNA
<213> mh7-19940740/773/856-F
<400> 163
ctttttggat tcatggacaa accaga 26
<210> 164
<211> 26
<212> DNA
<213> mh7-19940740/773/856-R
<400> 164
aggaaagcct gacctacaca aataat 26
<210> 165
<211> 27
<212> DNA
<213> mh7-35567404/461/536-F
<400> 165
tgtataaatt ctgtctgtgt gaccgaa 27
<210> 166
<211> 28
<212> DNA
<213> mh7-35567404/461/536-R
<400> 166
gacataacaa tttggagaaa ccagagac 28
<210> 167
<211> 21
<212> DNA
<213> mh7-44151710/747/763-F
<400> 167
catcccactg ccttgattca c 21
<210> 168
<211> 23
<212> DNA
<213> mh7-44151710/747/763-R
<400> 168
gacagcaatc acaatgtcta ccc 23
<210> 169
<211> 33
<212> DNA
<213> mh7-54459338/406/443/474-F
<400> 169
agatcactga agcttaaaac ttattaaaac atg 33
<210> 170
<211> 22
<212> DNA
<213> mh7-54459338/406/443/474-R
<400> 170
ttgggttagt gcatgcaagt ca 22
<210> 171
<211> 25
<212> DNA
<213> mh7-66228193/350/355-F
<400> 171
tgggaagtca ttagagggta tcaga 25
<210> 172
<211> 25
<212> DNA
<213> mh7-66228193/350/355-R
<400> 172
ctgatacatg tcagaatgca acagg 25
<210> 173
<211> 25
<212> DNA
<213> mh7-88765334/417/434-F
<400> 173
tggtatcatg ggtaggctaa atgtg 25
<210> 174
<211> 23
<212> DNA
<213> mh7-88765334/417/434-R
<400> 174
ccaacacagc ctaaaaccaa ctg 23
<210> 175
<211> 17
<212> DNA
<213> mh7-98286323/325/385/477-F
<400> 175
tgccagcccc ctctgtc 17
<210> 176
<211> 22
<212> DNA
<213> mh7-98286323/325/385/477-R
<400> 176
gagttggtga ggtgggtttc aa 22
<210> 177
<211> 30
<212> DNA
<213> mh7-109825096/124/233/285-F
<400> 177
caaaaataat catccaacag tgaagagaag 30
<210> 178
<211> 34
<212> DNA
<213> mh7-109825096/124/233/285-R
<400> 178
ggtaattttg tagagaaaat acaaactatt cttt 34
<210> 179
<211> 21
<212> DNA
<213> mh7-116223260/387/392-F
<400> 179
ggggcctaaa tttcgctcct a 21
<210> 180
<211> 24
<212> DNA
<213> mh7-116223260/387/392-R
<400> 180
atgccacagt catccttaaa gaac 24
<210> 181
<211> 21
<212> DNA
<213> mh7-128870866/888/957/970-F
<400> 181
gtgtggaagg tgcttcactg a 21
<210> 182
<211> 23
<212> DNA
<213> mh7-128870866/888/957/970-R
<400> 182
cttcctggct ctagtctagg tga 23
<210> 183
<211> 27
<212> DNA
<213> mh8-547113/127/247-F
<400> 183
catttagctt tcttctgaag aagagca 27
<210> 184
<211> 25
<212> DNA
<213> mh8-547113/127/247-R
<400> 184
gacactggat tgtgtaaagt atgcc 25
<210> 185
<211> 21
<212> DNA
<213> mh8-10518109/145/239-F
<400> 185
atcacaatgc agggcagagt g 21
<210> 186
<211> 22
<212> DNA
<213> mh8-10518109/145/239-R
<400> 186
acaccaaggc agacaaggag ta 22
<210> 187
<211> 22
<212> DNA
<213> mh8-19756502/636/649-F
<400> 187
ggcacagtgc tgttaacatc tc 22
<210> 188
<211> 18
<212> DNA
<213> mh8-19756502/636/649-R
<400> 188
gctagtgggg gatgtggc 18
<210> 189
<211> 34
<212> DNA
<213> mh8-29616927/7078/7103-F
<400> 189
ggaacttgta tttggttact taattttgtt tcag 34
<210> 190
<211> 23
<212> DNA
<213> mh8-29616927/7078/7103-R
<400> 190
agtcctgtgg gaatagcaca gat 23
<210> 191
<211> 22
<212> DNA
<213> mh8-60271584/624/690-F
<400> 191
gcaaggttgc caagaaacac tg 22
<210> 192
<211> 22
<212> DNA
<213> mh8-60271584/624/690-R
<400> 192
cactccagct gactacaagc aa 22
<210> 193
<211> 26
<212> DNA
<213> mh8-68082699/750/828-F
<400> 193
cagtcaaaac agcggtagtt tatagg 26
<210> 194
<211> 26
<212> DNA
<213> mh8-68082699/750/828-R
<400> 194
tctgtatcta catcaatgac acccaa 26
<210> 195
<211> 24
<212> DNA
<213> mh8-101133535/641/643/685-F
<400> 195
ggaaaaagtc cattcctcac ttgg 24
<210> 196
<211> 22
<212> DNA
<213> mh8-101133535/641/643/685-R
<400> 196
ctgaatctac acaaccgggt ga 22
<210> 197
<211> 22
<212> DNA
<213> mh8-116835749/847/860-F
<400> 197
tgtccatcgt ccttgaacac ag 22
<210> 198
<211> 22
<212> DNA
<213> mh8-116835749/847/860-R
<400> 198
ccttcaccag tgcctgtact tt 22
<210> 199
<211> 24
<212> DNA
<213> mh8-136290219/272/289-F
<400> 199
gaaagatgct ccctatttgg gaga 24
<210> 200
<211> 24
<212> DNA
<213> mh8-136290219/272/289-R
<400> 200
gactagggag aaccgaatag atgc 24
<210> 201
<211> 23
<212> DNA
<213> mh8-142661001/023/065-F
<400> 201
ggactttaac tggggacaac tga 23
<210> 202
<211> 23
<212> DNA
<213> mh8-142661001/023/065-R
<400> 202
ccatatgctt cattcgtgct cag 23
<210> 203
<211> 23
<212> DNA
<213> mh9-6000422/551/580-F
<400> 203
tgttctgggc aactctcatc tta 23
<210> 204
<211> 33
<212> DNA
<213> mh9-6000422/551/580-R
<400> 204
agaattaaga caataaaaat gtcctaacag att 33
<210> 205
<211> 24
<212> DNA
<213> mh9-23635143/146/207-F
<400> 205
gtctctggaa ggcatgtaaa actc 24
<210> 206
<211> 23
<212> DNA
<213> mh9-23635143/146/207-R
<400> 206
ctgaagttgc cgtgatatca ctc 23
<210> 207
<211> 31
<212> DNA
<213> mh9-36010365/433/555-F
<400> 207
agctgtttca ttggtatgat attattctca c 31
<210> 208
<211> 30
<212> DNA
<213> mh9-36010365/433/555-R
<400> 208
tgcagatcat atgaagaaaa tattccctct 30
<210> 209
<211> 21
<212> DNA
<213> mh9-88945871/6039/6042/6046/6056-F
<400> 209
gcacctgctc catcatgact g 21
<210> 210
<211> 25
<212> DNA
<213> mh9-88945871/6039/6042/6046/6056-R
<400> 210
caacatacac acagtagcca aagga 25
<210> 211
<211> 20
<212> DNA
<213> mh9-116564334/350/488-F
<400> 211
<210> 212
<211> 23
<212> DNA
<213> mh9-116564334/350/488-R
<400> 212
ggaattaatc atgtggccag cac 23
<210> 213
<211> 21
<212> DNA
<213> mh9-135392593/625/637/711-F
<400> 213
agaggcagca tccacaagtt g 21
<210> 214
<211> 21
<212> DNA
<213> mh9-135392593/625/637/711-R
<400> 214
acctggcaga tcactgaagg t 21
<210> 215
<211> 23
<212> DNA
<213> mh10-52144387/438/469-F
<400> 215
ggaaagctgg aagatgtgtt tgg 23
<210> 216
<211> 22
<212> DNA
<213> mh10-52144387/438/469-R
<400> 216
tgtgcctggc cctagactaa ag 22
<210> 217
<211> 22
<212> DNA
<213> mh10-62848538/544/601-F
<400> 217
tcccctatag ccacacatcc at 22
<210> 218
<211> 23
<212> DNA
<213> mh10-62848538/544/601-R
<400> 218
atgccaggta cttggtattt gct 23
<210> 219
<211> 19
<212> DNA
<213> mh10-71634144/299/303-F
<400> 219
ccaggcagct gcagattgg 19
<210> 220
<211> 22
<212> DNA
<213> mh10-71634144/299/303-R
<400> 220
gatgcacgga gaagagtcca aa 22
<210> 221
<211> 22
<212> DNA
<213> mh10-79630161/180/219-F
<400> 221
gcatctctca ctgcatcttg ga 22
<210> 222
<211> 21
<212> DNA
<213> mh10-79630161/180/219-R
<400> 222
tgagtctgga gggattcagc t 21
<210> 223
<211> 22
<212> DNA
<213> mh10-90206230/327/348-F
<400> 223
gaaatggcat ccgatccaca ag 22
<210> 224
<211> 22
<212> DNA
<213> mh10-90206230/327/348-R
<400> 224
ctgatcactg cctttgtggt tg 22
<210> 225
<211> 22
<212> DNA
<213> mh10-99523571/573/632-F
<400> 225
agggagaaag gaaactgctc tg 22
<210> 226
<211> 22
<212> DNA
<213> mh10-99523571/573/632-R
<400> 226
tctctactct gcgaattcct gc 22
<210> 227
<211> 21
<212> DNA
<213> mh10-111432667/700/772-F
<400> 227
ccccatgctg tttgtctgtc t 21
<210> 228
<211> 21
<212> DNA
<213> mh10-111432667/700/772-R
<400> 228
ctgtggcagc tgaaaccagt a 21
<210> 229
<211> 22
<212> DNA
<213> mh10-121111532/675/691-F
<400> 229
tgatcatgtc agaggtggct ca 22
<210> 230
<211> 23
<212> DNA
<213> mh10-121111532/675/691-R
<400> 230
gtcagggttg agaatgcaca tca 23
<210> 231
<211> 23
<212> DNA
<213> mh10-132237561/688/743-F
<400> 231
gtggtaacca ctccttccta agc 23
<210> 232
<211> 22
<212> DNA
<213> mh10-132237561/688/743-R
<400> 232
ctgattcttc cagggctggt tc 22
<210> 233
<211> 22
<212> DNA
<213> mh10-12081667/668/772-F
<400> 233
tgccaaaatc aaccccctct tc 22
<210> 234
<211> 22
<212> DNA
<213> mh10-12081667/668/772-R
<400> 234
acaggcctcc tcagaacttc ta 22
<210> 235
<211> 22
<212> DNA
<213> mh10-23068047/080/131-F
<400> 235
aaaagacacc ggtgttccac tg 22
<210> 236
<211> 22
<212> DNA
<213> mh10-23068047/080/131-R
<400> 236
aaggggcaaa aggaaactgg ag 22
<210> 237
<211> 23
<212> DNA
<213> mh10-35046057/116/252-F
<400> 237
ggaaaagcat ccaggcaatt acc 23
<210> 238
<211> 22
<212> DNA
<213> mh10-35046057/116/252-R
<400> 238
tctcaccaga ttgaatgtgg gc 22
<210> 239
<211> 22
<212> DNA
<213> mh10-43083331/467/505-F
<400> 239
cagcaccttg aaaactcgtg ac 22
<210> 240
<211> 23
<212> DNA
<213> mh10-43083331/467/505-R
<400> 240
ctcaaagctc cttaagcctc agg 23
<210> 241
<211> 21
<212> DNA
<213> mh11-12051115/117/276/279-F
<400> 241
ttggggaaga ggtttcctgg a 21
<210> 242
<211> 21
<212> DNA
<213> mh11-12051115/117/276/279-R
<400> 242
agcagctgag gaactctgag a 21
<210> 243
<211> 22
<212> DNA
<213> mh11-24408883/9048/9072-F
<400> 243
caggttgtac acaccatggc at 22
<210> 244
<211> 22
<212> DNA
<213> mh11-24408883/9048/9072-R
<400> 244
acagcactga aagtgtcaac gg 22
<210> 245
<211> 26
<212> DNA
<213> mh11-35239380/487/552-F
<400> 245
gaagctcttg gctaaagaaa tttcct 26
<210> 246
<211> 23
<212> DNA
<213> mh11-35239380/487/552-R
<400> 246
cctttgtgtg gttcatcaac gag 23
<210> 247
<211> 19
<212> DNA
<213> mh11-62787587/614/770-F
<400> 247
cgatgactcg gacccaggt 19
<210> 248
<211> 18
<212> DNA
<213> mh11-62787587/614/770-R
<400> 248
aggagccacg aaacccga 18
<210> 249
<211> 22
<212> DNA
<213> mh11-70104795/890/982-F
<400> 249
gaacccagag ggactttgga tg 22
<210> 250
<211> 21
<212> DNA
<213> mh11-70104795/890/982-R
<400> 250
caagcccctg ttctggatct g 21
<210> 251
<211> 28
<212> DNA
<213> mh11-80844932/5097/5104-F
<400> 251
actatatgct aacaagcaat ggtttgag 28
<210> 252
<211> 24
<212> DNA
<213> mh11-80844932/5097/5104-R
<400> 252
gtgagaaata tgctccaatc ccct 24
<210> 253
<211> 24
<212> DNA
<213> mh11-92548924/9009/9081-F
<400> 253
gaagtgagag aagttggcat ttgc 24
<210> 254
<211> 23
<212> DNA
<213> mh11-92548924/9009/9081-R
<400> 254
ccatttggca cccagaagaa gta 23
<210> 255
<211> 22
<212> DNA
<213> mh11-115165135/208/273-F
<400> 255
ggtgtccaca ctgacaattt gc 22
<210> 256
<211> 22
<212> DNA
<213> mh11-115165135/208/273-R
<400> 256
tgagcacgta gagcaagttc tg 22
<210> 257
<211> 22
<212> DNA
<213> mh11-127025586/628/687-F
<400> 257
gggaagcagg agaattgttt gg 22
<210> 258
<211> 22
<212> DNA
<213> mh11-127025586/628/687-R
<400> 258
ctctgaaacc agttcctcca ct 22
<210> 259
<211> 22
<212> DNA
<213> mh11-134478240/245/322-F
<400> 259
gctgattctc aaagtggcct ct 22
<210> 260
<211> 22
<212> DNA
<213> mh11-134478240/245/322-R
<400> 260
tggaaaggac ccttctttgc ag 22
<210> 261
<211> 22
<212> DNA
<213> mh12-13420587/700/702-F
<400> 261
ttgacactct gggccttaat gg 22
<210> 262
<211> 22
<212> DNA
<213> mh12-13420587/700/702-R
<400> 262
caacaaggtg agagtcacag gt 22
<210> 263
<211> 22
<212> DNA
<213> mh12-29086729/787/889-F
<400> 263
cagggacttt ttctgcacag ga 22
<210> 264
<211> 22
<212> DNA
<213> mh12-29086729/787/889-R
<400> 264
tagcagctca ggcagctaga ta 22
<210> 265
<211> 22
<212> DNA
<213> mh12-52741299/328/333/407/428-F
<400> 265
ggaggcagaa aggaaaacat gc 22
<210> 266
<211> 22
<212> DNA
<213> mh12-52741299/328/333/407/428-R
<400> 266
ctctgacgat ttgcatttgg gc 22
<210> 267
<211> 22
<212> DNA
<213> mh12-64611235/285/299-F
<400> 267
gcttgggtca acgtgtaata gc 22
<210> 268
<211> 20
<212> DNA
<213> mh12-64611235/285/299-R
<400> 268
<210> 269
<211> 24
<212> DNA
<213> mh12-78576646/704/730/815-F
<400> 269
ccactttagg accaggtcta aagg 24
<210> 270
<211> 28
<212> DNA
<213> mh12-78576646/704/730/815-R
<400> 270
gactttgatt tgaaccatgt atttgacc 28
<210> 271
<211> 23
<212> DNA
<213> mh12-93864450/453/506-F
<400> 271
gatgggaata ttagccccac aca 23
<210> 272
<211> 23
<212> DNA
<213> mh12-93864450/453/506-R
<400> 272
gagagctaca tttcaaggag gca 23
<210> 273
<211> 20
<212> DNA
<213> mh12-104583417/426/511/516/597-F
<400> 273
aatggggctc agctttgcat 20
<210> 274
<211> 22
<212> DNA
<213> mh12-104583417/426/511/516/597-R
<400> 274
catcaagaag tgctgaaggg gt 22
<210> 275
<211> 22
<212> DNA
<213> mh12-120495289/370/414-F
<400> 275
ccaaccaaga atctctccca ca 22
<210> 276
<211> 22
<212> DNA
<213> mh12-120495289/370/414-R
<400> 276
aaggagggag ggtgtttcct ta 22
<210> 277
<211> 22
<212> DNA
<213> mh13-24130689/693/782/792-F
<400> 277
tgctgataac atgctgacgg tt 22
<210> 278
<211> 22
<212> DNA
<213> mh13-24130689/693/782/792-R
<400> 278
tgtctcattc ctccgtagct ca 22
<210> 279
<211> 22
<212> DNA
<213> mh13-46330559/633/668-F
<400> 279
tcaccagacc agatacgagg tt 22
<210> 280
<211> 22
<212> DNA
<213> mh13-46330559/633/668-R
<400> 280
acgtccaagt acatgcacac ag 22
<210> 281
<211> 21
<212> DNA
<213> mh13-53365014/091/197-F
<400> 281
gcagtcctgg attgatcagc a 21
<210> 282
<211> 30
<212> DNA
<213> mh13-53365014/091/197-R
<400> 282
acaggagagt catcaattgt caattaaaag 30
<210> 283
<211> 24
<212> DNA
<213> mh13-75948926/8956/9019-F
<400> 283
caggaagccc atactatcaa caca 24
<210> 284
<211> 26
<212> DNA
<213> mh13-75948926/8956/9019-R
<400> 284
aacccgtctt agggcttata aattgt 26
<210> 285
<211> 25
<212> DNA
<213> mh13-86540463/512/659-F
<400> 285
gcttcgtgct agataaagcc ttatg 25
<210> 286
<211> 31
<212> DNA
<213> mh13-86540463/512/659-R
<400> 286
aaaaacacaa caatataaat gcattcatac a 31
<210> 287
<211> 23
<212> DNA
<213> mh13-96407596/649/753-F
<400> 287
gcgttcacct ggttagatag gtt 23
<210> 288
<211> 27
<212> DNA
<213> mh13-96407596/649/753-R
<400> 288
tccttttctt ttagcatttc ccttgtt 27
<210> 289
<211> 23
<212> DNA
<213> mh13-102654868/4972/5010-F
<400> 289
gctgtacaga tgacactgat gca 23
<210> 290
<211> 23
<212> DNA
<213> mh13-102654868/4972/5010-R
<400> 290
tgtcattctc cctctgaggt cag 23
<210> 291
<211> 24
<212> DNA
<213> mh13-114035629/633/707-F
<400> 291
ggtccctatc catctttctc agga 24
<210> 292
<211> 24
<212> DNA
<213> mh13-114035629/633/707-R
<400> 292
cacatctctc cagggttgta agag 24
<210> 293
<211> 23
<212> DNA
<213> mh14-20727667/710/808-F
<400> 293
agaagtaatc cttgctggac aca 23
<210> 294
<211> 23
<212> DNA
<213> mh14-20727667/710/808-R
<400> 294
ctagaacgtg aagaacccag tga 23
<210> 295
<211> 22
<212> DNA
<213> mh14-33572037/068/090/139-F
<400> 295
tgggctaatg caaaggacag ac 22
<210> 296
<211> 20
<212> DNA
<213> mh14-33572037/068/090/139-R
<400> 296
<210> 297
<211> 20
<212> DNA
<213> mh14-44538405/436/523-F
<400> 297
<210> 298
<211> 20
<212> DNA
<213> mh14-44538405/436/523-R
<400> 298
<210> 299
<211> 22
<212> DNA
<213> mh14-67511789/809/813/925-F
<400> 299
gtcgcctttc agaaccatcc tt 22
<210> 300
<211> 22
<212> DNA
<213> mh14-67511789/809/813/925-R
<400> 300
ggaaaaggca gtggttttca cc 22
<210> 301
<211> 24
<212> DNA
<213> mh14-80998510/557/611-F
<400> 301
catgtttggt gggtgttaca aagg 24
<210> 302
<211> 24
<212> DNA
<213> mh14-80998510/557/611-R
<400> 302
gagagacagc caagattagc catt 24
<210> 303
<211> 22
<212> DNA
<213> mh14-90301415/512/516-F
<400> 303
ctgtcgggtt tgaagaaggt ga 22
<210> 304
<211> 27
<212> DNA
<213> mh14-90301415/512/516-R
<400> 304
tggattataa tactgtgtga gacgctt 27
<210> 305
<211> 19
<212> DNA
<213> mh14-104316322/414/453-F
<400> 305
tggcccatct tctgggact 19
<210> 306
<211> 19
<212> DNA
<213> mh14-104316322/414/453-R
<400> 306
ccaacctgga agcacctgt 19
<210> 307
<211> 26
<212> DNA
<213> mh15-27602580/609/747-F
<400> 307
caaccacccc ttcacatttt atgttc 26
<210> 308
<211> 22
<212> DNA
<213> mh15-27602580/609/747-R
<400> 308
tctgtggggt tctttttgtt gt 22
<210> 309
<211> 23
<212> DNA
<213> mh15-53159314/324/431-F
<400> 309
tcactaaata tggtcccgca agg 23
<210> 310
<211> 22
<212> DNA
<213> mh15-53159314/324/431-R
<400> 310
agacaagggc agaatggtga ac 22
<210> 311
<211> 22
<212> DNA
<213> mh15-69304596/621/651-F
<400> 311
taattctcac ccatgccctg tc 22
<210> 312
<211> 22
<212> DNA
<213> mh15-69304596/621/651-R
<400> 312
ctgaccatct tggtcaaagg gt 22
<210> 313
<211> 20
<212> DNA
<213> mh15-78831543/604/674/697-F
<400> 313
<210> 314
<211> 17
<212> DNA
<213> mh15-78831543/604/674/697-R
<400> 314
agcagcagcg aacaggt 17
<210> 315
<211> 23
<212> DNA
<213> mh15-91536647/714/773-F
<400> 315
actccaagga ggacaaatgc tag 23
<210> 316
<211> 23
<212> DNA
<213> mh15-91536647/714/773-R
<400> 316
caagactgtg gcaggtgaat aga 23
<210> 317
<211> 22
<212> DNA
<213> mh15-101401241/328/414-F
<400> 317
ctagagacat tcggcagtgt ca 22
<210> 318
<211> 22
<212> DNA
<213> mh15-101401241/328/414-R
<400> 318
gcatctaagt ccacttgcct tg 22
<210> 319
<211> 18
<212> DNA
<213> mh16-828087/161/223/242-F
<400> 319
gggaacagcc cagctcag 18
<210> 320
<211> 21
<212> DNA
<213> mh16-828087/161/223/242-R
<400> 320
gttgtaggca ggtgtctgct t 21
<210> 321
<211> 27
<212> DNA
<213> mh16-19880935/0984/1051-F
<400> 321
ccattctaga acattcttac tggcctt 27
<210> 322
<211> 25
<212> DNA
<213> mh16-19880935/0984/1051-R
<400> 322
gagagtgaac tgtgtacccg tattc 25
<210> 323
<211> 21
<212> DNA
<213> mh16-28499072/134/216-F
<400> 323
gcggggataa cacaaacctc t 21
<210> 324
<211> 22
<212> DNA
<213> mh16-28499072/134/216-R
<400> 324
aacccctctc tgctcctaca tt 22
<210> 325
<211> 22
<212> DNA
<213> mh16-49595295/400/414-F
<400> 325
ttttcctccc caatgaggac ag 22
<210> 326
<211> 21
<212> DNA
<213> mh16-49595295/400/414-R
<400> 326
ggaaggcagg aagatgctag t 21
<210> 327
<211> 22
<212> DNA
<213> mh16-57674122/144/239-F
<400> 327
aagcgcaggg agagtatagt ca 22
<210> 328
<211> 22
<212> DNA
<213> mh16-57674122/144/239-R
<400> 328
tctccccaat ccatctctcc tt 22
<210> 329
<211> 23
<212> DNA
<213> mh16-76868825/950/953-F
<400> 329
acacagaaac actgctcaga gtc 23
<210> 330
<211> 26
<212> DNA
<213> mh16-76868825/950/953-R
<400> 330
acgtatagtt ttcacagtga tgacct 26
<210> 331
<211> 22
<212> DNA
<213> mh17-12218325/392/434/522-F
<400> 331
atgtgtggtg aagagaggat gg 22
<210> 332
<211> 22
<212> DNA
<213> mh17-12218325/392/434/522-R
<400> 332
cctttgcgaa actgcaaaaa cc 22
<210> 333
<211> 20
<212> DNA
<213> mh17-33970547/587/589/732-F
<400> 333
<210> 334
<211> 21
<212> DNA
<213> mh17-33970547/587/589/732-R
<400> 334
gagtcagcct ggacaccttt c 21
<210> 335
<211> 26
<212> DNA
<213> mh17-40705403/505/528/533-F
<400> 335
ggtgcttcca gaaatagttt gtatcc 26
<210> 336
<211> 25
<212> DNA
<213> mh17-40705403/505/528/533-R
<400> 336
actcctgtga aaacagtaca ggtag 25
<210> 337
<211> 34
<212> DNA
<213> mh17-54617721/723/870-F
<400> 337
gatcaaatgt ctttatataa gaaaaacaga gttc 34
<210> 338
<211> 23
<212> DNA
<213> mh17-54617721/723/870-R
<400> 338
cagaatctca gaagttccct ggt 23
<210> 339
<211> 20
<212> DNA
<213> mh17-81067161/204/334-F
<400> 339
<210> 340
<211> 20
<212> DNA
<213> mh17-81067161/204/334-R
<400> 340
<210> 341
<211> 24
<212> DNA
<213> mh18-1648913/8946/9009-F
<400> 341
ggaaatgtac atgggaggag gaag 24
<210> 342
<211> 26
<212> DNA
<213> mh18-1648913/8946/9009-R
<400> 342
cgatgaagaa aattgtagcc ctaagc 26
<210> 343
<211> 22
<212> DNA
<213> mh18-11578129/218/238-F
<400> 343
aaatcatccc caccagaacc at 22
<210> 344
<211> 22
<212> DNA
<213> mh18-11578129/218/238-R
<400> 344
gcatgcatgt ggctttcatt tc 22
<210> 345
<211> 27
<212> DNA
<213> mh18-42056338/401/516-F
<400> 345
gttgtttgag ttttctgaca agaagga 27
<210> 346
<211> 27
<212> DNA
<213> mh18-42056338/401/516-R
<400> 346
acaggtgtag atacattttc cttggtt 27
<210> 347
<211> 22
<212> DNA
<213> mh18-52103231/255/320-F
<400> 347
tctcctgggc atccatttat gc 22
<210> 348
<211> 22
<212> DNA
<213> mh18-52103231/255/320-R
<400> 348
accaggctac ccaaatcatc ac 22
<210> 349
<211> 22
<212> DNA
<213> mh18-78243467/550/557/627-F
<400> 349
cccgattctg agcatcaagg aa 22
<210> 350
<211> 25
<212> DNA
<213> mh18-78243467/550/557/627-R
<400> 350
caagcataca tctggaggac aaaac 25
<210> 351
<211> 21
<212> DNA
<213> mh19-645780/797/801/903/934-F
<400> 351
gcttattcgc aagcccaaac c 21
<210> 352
<211> 17
<212> DNA
<213> mh19-645780/797/801/903/934-R
<400> 352
aggagcctgc tgcacga 17
<210> 353
<211> 22
<212> DNA
<213> mh19-32967227/289/331-F
<400> 353
acattgcctc acacctcata gg 22
<210> 354
<211> 22
<212> DNA
<213> mh19-32967227/289/331-R
<400> 354
ctcaccactt tggggatgaa ga 22
<210> 355
<211> 22
<212> DNA
<213> mh19-55911606/667/708-F
<400> 355
agttagagct gctccagacc at 22
<210> 356
<211> 22
<212> DNA
<213> mh19-55911606/667/708-R
<400> 356
agcagtcaca tccttgaaag gg 22
<210> 357
<211> 22
<212> DNA
<213> mh20-4334692/856/885-F
<400> 357
tcaccctaaa acctacccgg aa 22
<210> 358
<211> 21
<212> DNA
<213> mh20-4334692/856/885-R
<400> 358
acagtgcgct tctcttcaga c 21
<210> 359
<211> 21
<212> DNA
<213> mh20-12674881/4924/5006-F
<400> 359
gtgggctact gtgtatgcag a 21
<210> 360
<211> 22
<212> DNA
<213> mh20-12674881/4924/5006-R
<400> 360
gagaggtacc aagtgtggac ag 22
<210> 361
<211> 20
<212> DNA
<213> mh20-22330802/810/874/973-F
<400> 361
<210> 362
<211> 24
<212> DNA
<213> mh20-22330802/810/874/973-R
<400> 362
acaggacagt agaagatccc tacc 24
<210> 363
<211> 23
<212> DNA
<213> mh20-40717241/300/307-F
<400> 363
tggctggcat ttcttagagg atg 23
<210> 364
<211> 21
<212> DNA
<213> mh20-40717241/300/307-R
<400> 364
ctcttgggcg cctttattcc t 21
<210> 365
<211> 20
<212> DNA
<213> mh20-54160200/230/378-F
<400> 365
<210> 366
<211> 20
<212> DNA
<213> mh20-54160200/230/378-R
<400> 366
<210> 367
<211> 21
<212> DNA
<213> mh20-63124779/838/974-F
<400> 367
agaggaggga atggaggtga g 21
<210> 368
<211> 21
<212> DNA
<213> mh20-63124779/838/974-R
<400> 368
gttcatcctg acagccctca g 21
<210> 369
<211> 29
<212> DNA
<213> mh21-18775368/420/523-F
<400> 369
ggacttaaaa agtaaaacta ggctttgtg 29
<210> 370
<211> 26
<212> DNA
<213> mh21-18775368/420/523-R
<400> 370
agcaggaagt ttgaacaaaa tggtta 26
<210> 371
<211> 29
<212> DNA
<213> mh21-30568687/804/813-F
<400> 371
ctgtatcagt ggtattccag aaaatatcc 29
<210> 372
<211> 29
<212> DNA
<213> mh21-30568687/804/813-R
<400> 372
ccatcccata tcaacatttt aaaggagaa 29
<210> 373
<211> 18
<212> DNA
<213> mh21-38685564/617/643/673-F
<400> 373
tgtggctccc caagtcca 18
<210> 374
<211> 18
<212> DNA
<213> mh21-38685564/617/643/673-R
<400> 374
gcatggtggc tccttgct 18
<210> 375
<211> 18
<212> DNA
<213> mh21-46138559/672/677-F
<400> 375
caggccaggt tcccacac 18
<210> 376
<211> 19
<212> DNA
<213> mh21-46138559/672/677-R
<400> 376
gtacctgggt gctgtgacg 19
<210> 377
<211> 23
<212> DNA
<213> mh22-22132253/415/434-F
<400> 377
accccatttc ctcatacatt gct 23
<210> 378
<211> 27
<212> DNA
<213> mh22-22132253/415/434-R
<400> 378
gtctgtccta ttatgtagcc ctttact 27
<210> 379
<211> 23
<212> DNA
<213> mh22-38109259/340/349-F
<400> 379
aggggaggaa aaaggtgtag aga 23
<210> 380
<211> 23
<212> DNA
<213> mh22-38109259/340/349-R
<400> 380
agcttagaac aggaccccaa aag 23
<210> 381
<211> 22
<212> DNA
<213> mh22-48867296/365/380-F
<400> 381
gagcactgtg atcaaaggct ca 22
<210> 382
<211> 22
<212> DNA
<213> mh22-48867296/365/380-R
<400> 382
cacattctgt tgctggaagg ga 22
<210> 383
<211> 24
<212> DNA
<213> rs2056688-F
<400> 383
ggatcatgat acctgcccaa catg 24
<210> 384
<211> 22
<212> DNA
<213> rs2056688-R
<400> 384
accacacgtg ttcacaccta ga 22
<210> 385
<211> 22
<212> DNA
<213> rs1534285-F
<400> 385
ccacaagact aagccaagac ca 22
<210> 386
<211> 22
<212> DNA
<213> rs1534285-R
<400> 386
acaggacgtc aattcaccag aa 22
<210> 387
<211> 21
<212> DNA
<213> rs763056-F
<400> 387
gaaagtcatc caccccagca t 21
<210> 388
<211> 21
<212> DNA
<213> rs763056-R
<400> 388
tacctgacct cctcttggca a 21
<210> 389
<211> 22
<212> DNA
<213> rs17316592-F
<400> 389
ccttctgtgt ttgtccagag gt 22
<210> 390
<211> 22
<212> DNA
<213> rs17316592-R
<400> 390
gttgccatct gtcccctctt aa 22
<210> 391
<211> 23
<212> DNA
<213> rs3900-F
<400> 391
ggaccctgaa atacagaact gca 23
<210> 392
<211> 26
<212> DNA
<213> rs3900-R
<400> 392
ctcatttttg aagctcgtga aacaga 26
<210> 393
<211> 22
<212> DNA
<213> rs13447352-F
<400> 393
tagagggtat tggggtaggc aa 22
<210> 394
<211> 22
<212> DNA
<213> rs13447352-R
<400> 394
atgccttcag gcttctagct tc 22
Claims (10)
1. A primer set for amplifying genetic markers of 191 human micro-haplotype loci, characterized in that: the nucleotide sequence of the primer is as follows:
1）SEQ ID NO.1-2；2）SEQ ID NO.3-4；3）SEQ ID NO.5-6；4）SEQ ID NO.7-8；5）SEQ ID NO.9-10；6）SEQ ID NO.11-12；7）SEQ ID NO.13-14；8）SEQ ID NO.15-16；9）SEQ ID NO.17-18；10）SEQ ID NO.19-20；11）SEQ ID NO.21-22；12）SEQ ID NO.23-24；13）SEQ ID NO.25-26；14）SEQ ID NO.27-28；15）SEQ ID NO.29-30；16）SEQ ID NO.31-32；17）SEQ ID NO.33-34；18）SEQ ID NO.35-36；19）SEQ ID NO.37-38；20）SEQ ID NO.39-40；21）SEQ ID NO.41-42；22）SEQ ID NO.43-44；23）SEQ ID NO.45-46；24）SEQ ID NO.47-48；25）SEQ ID NO.49-50；26）SEQ ID NO.51-52；27）SEQ ID NO.53-54；28）SEQ ID NO.55-56；29）SEQ ID NO.57-58；30）SEQ ID NO.59-60；31）SEQ ID NO.61-62；32）SEQ ID NO.63-64；33）SEQ ID NO.65-66；34）SEQ ID NO.67-68；35）SEQ ID NO.69-70；36）SEQ ID NO.71-72；37）SEQ ID NO.73-74；38）SEQ ID NO.75-76；39）SEQ ID NO.77-78；40）SEQ ID NO.79-80；41）SEQ ID NO.81-82；42）SEQ ID NO.83-84；43）SEQ ID NO.85-86；44）SEQ ID NO.87-88；45）SEQ ID NO.89-90；46）SEQ ID NO.91-92；47）SEQ ID NO.93-94；48）SEQ ID NO.95-96；49）SEQ ID NO.97-98；50）SEQ ID NO.99-100；51）SEQ ID NO.101-102；52）SEQ ID NO.103-104；53）SEQ ID NO.105-106；54）SEQ ID NO.107-108；55）SEQ ID NO.109-110；56）SEQ ID NO.111-112；57）SEQ ID NO.113-114；58）SEQ ID NO.115-116；59）SEQ ID NO.117-118；60）SEQ ID NO.119-120；61）SEQ ID NO.121-122；62）SEQ ID NO.123-124；63）SEQ ID NO.125-126；64）SEQ ID NO.127-128；65）SEQ ID NO.129-130；66）SEQ ID NO.131-132；67）SEQ ID NO.133-134；68）SEQ ID NO.135-136；69）SEQ ID NO.137-138；70）SEQ ID NO.139-140；71）SEQ ID NO.141-142；72）SEQ ID NO.143-144；73）SEQ ID NO.145-146；74）SEQ ID NO.147-148；75）SEQ ID NO.149-150；76）SEQ ID NO.151-152；77）SEQ ID NO.153-154；78）SEQ ID NO.155-156；79）SEQ ID NO.157-158；80）SEQ ID NO.159-160；81）SEQ ID NO.161-162；82）SEQ ID NO.163-164；83）SEQ ID NO.165-166；84）SEQ ID NO.167-168；85）SEQ ID NO.169-170；86）SEQ ID NO.171-172；87）SEQ ID NO.173-174；88）SEQ ID NO.175-176；89）SEQ ID NO.177-178；90）SEQ ID NO.179-180；91）SEQ ID NO.181-182；92）SEQ ID NO.183-184；93）SEQ ID NO.185-186；94）SEQ ID NO.187-188；95）SEQ ID NO.189-190；96）SEQ ID NO.191-192；97）SEQ ID NO.193-194；98）SEQ ID NO.195-196；99）SEQ ID NO.197-198；100）SEQ ID NO.199-200；101）SEQ ID NO.201-202；102）SEQ ID NO.203-204；103）SEQ ID NO.205-206；104）SEQ ID NO.207-208；105）SEQ ID NO.209-210；106）SEQ ID NO.211-212；107）SEQ ID NO.213-214；108）SEQ ID NO.215-216；109）SEQ ID NO.217-218；110）SEQ ID NO.219-220；111）SEQ ID NO.221-222；112）SEQ ID NO.223-224；113）SEQ ID NO.225-226；114）SEQ ID NO.227-228；115）SEQ ID NO.229-230；116）SEQ ID NO.231-232；117）SEQ ID NO.233-234；118）SEQ ID NO.235-236；119）SEQ ID NO.237-238；120）SEQ ID NO.239-240；121）SEQ ID NO.241-242；122）SEQ ID NO.243-244；123）SEQ ID NO.245-246；124）SEQ ID NO.247-248；125）SEQ ID NO.249-250；126）SEQ ID NO.251-252；127）SEQ ID NO.253-254；128）SEQ ID NO.255-256；129）SEQ ID NO.257-258；130）SEQ ID NO.259-260；131）SEQ ID NO.261-262；132）SEQ ID NO.263-264；133）SEQ ID NO.265-266；134）SEQ ID NO.267-268；135）SEQ ID NO.269-270；136）SEQ ID NO.271-272；137）SEQ ID NO.273-274；138）SEQ ID NO.275-276；139）SEQ ID NO.277-278；140）SEQ ID NO.279-280；141）SEQ ID NO.281-282；142）SEQ ID NO.283-284；143）SEQ ID NO.285-286；144）SEQ ID NO.287-288；145）SEQ ID NO.289-290；146）SEQ ID NO.291-292；147）SEQ ID NO.293-294；148）SEQ ID NO.295-296；149）SEQ ID NO.297-298；150）SEQ ID NO.299-300；151）SEQ ID NO.301-302；152）SEQ ID NO.303-304；153）SEQ ID NO.305-306；154）SEQ ID NO.307-308；155）SEQ ID NO.309-310；156）SEQ ID NO.311-312；157）SEQ ID NO.313-314；158）SEQ ID NO.315-316；159）SEQ ID NO.317-318；160）SEQ ID NO.319-320；161）SEQ ID NO.321-322；162）SEQ ID NO.323-324；163）SEQ ID NO.325-326；164）SEQ ID NO.327-328；165）SEQ ID NO.329-330；166）SEQ ID NO.331-332；167）SEQ ID NO.333-334；168）SEQ ID NO.335-336；169）SEQ ID NO.337-338；170）SEQ ID NO.339-340；171）SEQ ID NO.341-342；172）SEQ ID NO.343-344；173）SEQ ID NO.345-346；174）SEQ ID NO.347-348；175）SEQ ID NO.349-350；176）SEQ ID NO.351-352；177）SEQ ID NO.353-354；178）SEQ ID NO.355-356；179）SEQ ID NO.357-358；180）SEQ ID NO.359-360；181）SEQ ID NO.361-362；182）SEQ ID NO.363-364；183）SEQ ID NO.365-366；184）SEQ ID NO.367-368；185）SEQ ID NO.369-370；186）SEQ ID NO.371-372；187）SEQ ID NO.373-374；188）SEQ ID NO.375-376；189）SEQ ID NO.377-378；190）SEQ ID NO.379-380；191）SEQ ID NO.381-382。
2. use of a primer combination for amplifying genetic markers of 191 human micro-haplotype loci according to claim 1 for forensic identification for non-diagnostic purposes.
3. Use of a primer combination for amplifying genetic markers of 191 human micro-haplotype loci according to claim 1 for identification of individuals of non-diagnostic interest.
4. Use of a primer combination for amplifying genetic markers of 191 human micro-haplotype loci according to claim 1 for genetic relationship identification for non-diagnostic purposes.
5. Use of a primer combination for amplifying genetic markers of 191 human micro-haplotype loci according to claim 4 for genetic relationship identification for non-diagnostic purposes, wherein: the genetic relationship identification comprises paternity identification, isotactic cell identification and two-level genetic relationship identification with only two individuals participating in.
6. A forensic identification kit, characterized in that: a primer combination comprising the genetic markers for amplifying 191 human microsloid loci of claim 1.
7. The forensic identification kit of claim 6, wherein: also included are buffer solutions, DNA polymerase and dNTPs.
8. The method of using a forensic identification kit for non-diagnostic purposes according to claim 6 wherein: extracting genome DNA of a sample to be detected as a template, carrying out PCR amplification by using the kit, carrying out a linker sequence PCR reaction on the obtained amplification product to obtain a multiple PCR library, and carrying out quantitative and second-generation sequencing detection analysis on the multiple PCR library to obtain a typing result of the micro-haplotype locus.
9. The method of using a forensic identification kit for non-diagnostic purposes according to claim 8 in which: the PCR amplification system comprises the following reagents and the dosage:
the PCR amplification procedure was:
the system for PCR reaction of the linker sequence comprises the following reagents and the dosage:
the PCR reaction of the linker sequence is as follows:
10. the method of using a forensic identification kit for non-diagnostic purposes according to claim 9 wherein: the dosage of the genome DNA is 5ng-20ng;
and/or said amplifying 191 a mixture of primers genetically tagged to a human microsloid locus, each primer at a concentration of 5-10 μm.
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