CN106399563A - Multi-locus STR analysis method - Google Patents
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Abstract
The invention discloses a multi-locus STR analysis method. According to the method, a single-chain DNA segment with a specific length is selectively chemically linked to different fluorescently-labeled STR segments, so that the number of STR segments which can be labeled by a single fluorescent dye can be increased twice or even more; the problem that the affiliation (namely genetic typing) of loca of labeled segments cannot be determined because each specific dye is difficult to confirm during the detection due to the relatively serious crossing of fluorescence spectra among the dyes when more STR segments are labeled by virtue of multi-color dyes (more than 5 colors) is solved.
Description
Technical field
The present invention relates to DNA fluorescent labeling and limited loci gene small fragment detection technique field are and in particular to one kind
The chemical linking techniques of DNA fragmentation and the fluorescence spectrum collection by capillary electrophoresis and analytical technology.
Background technology
Completing with the Human Genome Project, the mankind are unprecedented to having understood and grasped of itself hereditary information
Progressive.DNA detection technology has obtained fast development, and DNA detection efficiency improves constantly.DNA detection technology is in individual identification, wrecked
Person's identification, paternity test, blood relationship identification, criminal case detection, food and seed safety, medical diagnosis on disease, precisely medical treatment etc.
Aspect plays highly important effect.
Everyone DNA is different, therefore theoretically, can determine everyone unique body by full genome sequencing
Part.But the DNA of human body comprises nearly 3,000,000,000 bases, and between different people, more than 99.5% DNA sequence is identical, because
This, be sequenced by full genome and determine that the way of personal identification can take time and effort, and be completely unnecessary it is only necessary to exist to the mankind
Exist and detection is carried out on the locus of difference screen.Currently commercially way is by the STR fragment under limited loci
Detect and to set up everyone DNA data base, for identifying personal identification.
Identity authentication technology is also commonly known as genescan technology, and its principle is as follows：Normal human has 23 pairs of chromosomes, altogether
Article 46, chromosome.Every item chromosome is made up of two double-stranded DNAs, and each double-stranded DNA is single-stranded by two base complementrities
DNA is constituted.A lot of coding regions (i.e. gene regions) and noncoding region are had on each single stranded DNA, they are with linear alternative form
It is arranged on DNA.Typically one section of these noncoding regions are repeatedly weighed with certain short base sequence (typically in 2 to 6 bases)
Appear again existing DNA fragmentation, referred to as short sequence tandem repeat unit (Short Tandem Repeats, STR).Substantial amounts of research is sent out
Existing, the STR in each noncoding region assumes polymorphism, and that is, STR in same noncoding region for the different bions repeats
Number may be different, and in different noncoding regions, the base sequence of STR repetitives and repetition number are also to be completely independent
, do not associate each other.Therefore, the DNA sequence in these noncoding regions is used to distinguish different biology individual (inclusion mankind's basis
Body) excellent object.For example, if there are 10 kinds of polymorphisms (i.e. 10 different repetition number) under locus, then
Choose 10 locus, have 10 in theory10=100 hundred million kinds of polymorphisms.Therefore pass through to detect the STR piece in multiple noncoding regions
Section is so that it may distinguish everyone unique identity.Britain adopts 11 locus at present, and the U.S. adopts 13 locus, and Chinese
Using 18-22 locus.But, under many circumstances, so many locus numbers are also not enough to determine the body of a people
Part or feature are it is necessary to increase more locus to carry out more accurately and reliably identity authentication.
The STR polymorphism of limited loci can (wherein forward primer be special with certain by well-designed specifically two-way primer
Fixed dye marker), realize the short sequence in noncoding region comprising repetitives on DNA profiling chain is replicated through round pcr
And amplification, recycle capillary electrophoresis and detection technique of fluorescence to determine repetitives number and the locus ownership of each fragment, that is,
Gene type.
Multicolored fluorescent dye is usually used in the market to realize genescan, the DNA piece that different test kits are detected
Segment length scope is slightly changed, substantially between 80-350bp.Wherein, the different piece of a kind of one group of length of fluorochrome label
Section, as internal standard (determining the scale of DNA length), remaining four kinds of dyestuff is respectively intended to the multiple locus of labelling.When using four colors
It is clear that multiple locus must share same dyestuff carrys out labelling during 18 locus of dye marker.Locus ownership is basis
Color and two parameters of fragment length are determining.Therefore, in order to determine the different fragments base under same dye marker
Because of the ownership of seat, these fragment lengths must be well-designed in advance, allows the STR fragment under different genes seat between 80-350bp
Respectively occupy a specifically interval, overlap can not in length range each other.In real work, a lot of samples adopt general examination
The detection and analysis of agent box can't be made it is necessary to design has the test kit of more limited loci number when accurately judging, by expanding
The detection range of big DNA fragmentation length is to accommodate more STR fragments or to increase more fluorescent dyes realizations to increased gene
Seat is marked.In some cases, for example, when in criminal offense, biological sample is highly degraded, the DNA profiling chain of sample occurs
Fracture, its corresponding complete genome seat STR fragment is just difficult to replicate by round pcr, thus not only causes related gene seat
Information is lost, and unsuccessfully can only produce less fragment because replicating, and these fragments are overlapped with other small fragment areas, seriously
Interference detection results.Therefore, it is subject to by the strategy that the detection range of expansion DNA fragmentation length accommodates more STR fragments
Greatly limit, many methods that labelling more limited loci is come using increase fluorescent dye on market.The five of currently commercially use
Color dye system is difficult to realize the fluorescent labeling to more limited loci and sequence length within the narrow range of 80-350bp
Not cross arrangement.
It is clear that the application of this five-color system has larger limitation.It is in particular in：(1) locus number
Insufficient.In the case of Multi-example mixing, make not necessarily can accurately examine during genescan using less locus number
Go out the corresponding single identity of each component in biased sample.Especially when sample occurs degraded, the STR fragment of some locus because
Can not be expanded out by round pcr and invalid, then can serve as identifying that the effective gene seat STR number of identity is greatly reduced,
Thus accurate identity authentication cannot be made；(2) because a kind of dyestuff has been used for labelling internal standard, only four kinds of fluorescent dye deficiencies
With for labelling more limited loci.In the case of height of specimen degraded, because DNA profiling fracture is imperfect, by round pcr
It is difficult to replicate and expand larger fragment.Therefore on the biological sample of height degraded, widely used moonlet or microsatellite piece
The analytic process of section, tries one's best to the STR fragment of each locus and selects sequence the shortest, can not because of the degraded of template fundamental chain with reduction
Risk by required each locus STR fragment of PCR amplification；But, in a limited DNA fragmentation length range,
The locus number of homogencous dyes labelling will be extremely restricted.
Content of the invention
It is an object of the invention to provide a kind of limited loci STR analysis method, the inventive method is for multicolored fluorescence dye
The limitation when limited loci STR detects for the material, not only can extend single fluorescent dye can labelling locus STR fragment
Number, and can solve the problem that polychrome (more than 5 colors) dyestuff is led because of more serious fluorescence spectrum cross-cutting issue in labelling STR fragment
Cause during instrument detection, to be difficult to determine the fragment gene seat ownership of each dye marker.
The ultimate principle of the present invention is as follows：In each locus, repeated STR length is with high polymorphism
, it is the basis of modern identity authentication.Only comprising repeated STR unit is very little in itself.In order to make capillary tube electric
Swimming can analyze multiple STR fragments simultaneously, and these fragments must have difference on fragment length in base number, could be by area
Point.Therefore when selecting the STR length of multiple locus, have and be intended to some bases of multiselect outside STR repetitives, cause each base
Because being unlikely to occur base number identical between the STR fragment of seat.In some cases, for example in the biological sample of height degraded,
The DNA fragmentation replicating length by round pcr is extremely difficult, reduces the STR fragment of locus as far as possible, that is, adopt microsatellite fragment,
It is to realize the key that sample is successfully detected.The present invention is by pre-synthesis one section of sequence with test sample is unrelated, length is special
Fixed Single-stranded DNA fragments, it is right with numerous little STR Piece Selection ground then to be realized by special efficient chemical connection process
Connect, allow fragment that may be overlapping spatial displacement occur thus can be separated by electrophoresis.It is emphasized that due to connecting length used
Spending specific Single-stranded DNA fragments does not have any association with sample, although therefore sample fundamental chain ruptures because degrading,
The feature STR fragment that fragment length is different and comprises each locus still can be prepared by the method for the present invention.
The present invention do not change in the market commonly use DNA fragmentation length detection scope on the premise of, can be allowed each
Dyestuff respectively labelling one times of locus STR fragment more than original test kit, during design, newly-increased labeled locus are compiled to
One group, make each locus STR fragment in group not overlapping mutually, and without considering and homochromy marker gene seat fragment length originally
Whether overlapping problem.To newly-increased locus, need to introduce a foot carrying three functional groups between dyestuff and primer
Hand cradle small molecule, Liang Ge functional group therein constitutes basic framework with DNA backbone, for the connection between primer and dyestuff, the
Three are then connected chemically for the specific Single-stranded DNA fragments of length external with a section.
When, after PCR amplification and purification, one group of newly-increased fragment can be with the pre-synthesis specific DNA fragmentation of length
Chemical reaction is occurred to link together, the DNA fragmentation length after connection will increase, and common without scaffold molecule modification
One group of method labelling because reaction can not be connected chemically, its fragment length will not change.Therefore, use new method labelling
Newly-increased locus can be kept completely separate in electrophoresis with one group of locus of former commonsense method labelling, thus realize gene dividing
Type.
In the case of multicolor fluorescence (more than 5 colors) dye marker limited loci STR fragment, due to the fluorescence between dyestuff
Spectral cross problem, when fragment length is similar, spectrum each other severe jamming it is difficult to accurately differentiate the dyestuff of respective institute labelling,
Thus gene type can become highly difficult.The method of this patent is by above-mentioned class by cross one another for spectrum dye marker fragment
As method realize displaced from one another, spatially not overlapping, thus realize spectral cross minimizing, being beneficial to fluoroscopic examination
And gene type.
For reaching above-mentioned purpose, the present invention adopts the following technical scheme that：
A kind of limited loci STR analysis method, the method is included using by the primer pair polygenes of fluorochrome label
Seat STR fragment enter performing PCR replicate and amplification after specific for length Single-stranded DNA fragments can be selectively connected to expand
On some DNA fragmentation products after increasing.
Preferably, described is forward primer or reverse primer in two-way primer by the primer of fluorochrome label, and
Before fluorochrome label, a scaffold molecule is introduced on partly described forward primer or partly described reverse primer.
Preferably, described scaffold molecule includes a basic framework and one and is located at changing on described basic framework
Learn the functional group connecting.
Preferably, described fluorescent dye, scaffold molecule and described forward primer or described fluorescent dye, scaffold molecule
Spacer molecule can be provided with described reverse primer between any two；In same limited loci STR fragment, described spacer molecule
Structure can be identical or different, the structure of described spacer molecule includes linear structure or bifurcation structure.
Preferably, described functional group be maleimide, haloacetyl, amino, sulfydryl, azido, alkynyl, diazanyl,
At least one in azyloxy, aldehyde radical or ketone group.
Preferably, described amino also includes being changed the sulfydryl of generation further by amino by chemical conversioning reaction；Described
Chemical conversioning reaction includes and Traut reagent or anti-with the chemistry that N- acetyl group-homocysteinic acid thiolactone reagent occurs
Should.
Preferably, described maleimide has the structure as shown in formula (6) or formula (7),
In formula (7), R is straight chained alkyl；
Described aldehyde radical or ketone group have the structure as shown in formula (14),
In formula (14), R1=H or straight chained alkyl, R2For straight chained alkyl or alkoxyl.
Preferably, described basic framework includes thering is the structure shown in formula (1) or (2),
In formula (1) and (2), n=1,2,3,4, R is the functional group on described skeleton；
Or there is the structure shown in formula (3),
In formula (3), n=1,2,3, R is the functional group on described skeleton；
Or there is the structure shown in formula (4),
In formula (4), n=1,2；
Or there is the structure shown in formula (5),
Preferably, described Single-stranded DNA fragments are primers by chemosynthesis band particular functional group and biosynthesiss PCR is multiple
The specific fragment of a segment length that system amplification obtains, its sequence is generally unrelated with sample, and its length and sequence can be according to inspections
Survey requirement to adjust and change.
Beneficial effects of the present invention are as follows：
(1) this method can by single fluorescent dye can labelling STR segment number expand be twice even more many；
(2) solve the problems, such as to lead to because there being more serious fluorescence cross during polychrome (more than 5 colors) dye marker STR fragment
It is difficult to determine fragment home (gene type) problem of each dye marker during instrument detection.
By the spectral cross of each dyestuff, the impact to detection is reduced to minimum to the inventive method.Therefore, the present invention will be to future
STR detection in need to increase more limited loci provide a uniqueness and effective solution it is expected to become DNA from now on
A powerful technology platform in Examination and diagnosis.
Brief description
Fig. 1 is the mentality of designing schematic diagram of the present invention；
Fig. 2 a is the primer of chemosynthesis fluorochrome label and synthesizes STR fragment using round pcr；
Fig. 2 b is to realize, using displacement method, the principle schematic that dyestuff is capable of labelling more multiple clips；
Fig. 2 c is dye fluorescence spectral cross schematic diagram；
Fig. 3 is the layout strategy schematic diagram of the DNA primer of dye marker equal length of four color fluorescence cross；
Fig. 4 is the application schematic diagram of the inventive method；
Fig. 5 is the application schematic diagram of the inventive method；
Fig. 6 is spacer molecule and dyestuff, scaffold molecule, the connection schematic diagram of primer.
Specific embodiment
In order to be illustrated more clearly that the present invention, with reference to preferred embodiment, the present invention is described further.Ability
Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, and should not limit this with this
The protection domain of invention.
The present invention is directed to the limitation based on multicolored fluorescent labeling limited loci STR detection system, originally proposes profit
With Chemical ligation, the DNA fragmentation of one length-specific is selectively connected in different fluorescently-labeled STR fragments, thus
Reach two purposes：(1) single fluorescent dye is capable of the STR segments of labelling and can be extended one times of even more times；(2) when many
Color dyestuff (more than 5 colors) be used for labelling more STR fragment when, can because of dyestuff between more serious fluorescence spectrum cross-cutting issue and in quilt
It is difficult to during detection determine each specific dyestuff, thus not can determine that locus ownership (i.e. gene type) of institute's labeled fragment；
This method realizes sequence length displacement on identification spatial arrangement by being connected chemically, thus the dye marker piece by spectral cross
Section dislocation, is no longer detected simultaneously, thus can accurately determine the dyestuff used by fragment label.
The present invention utilizes chemical connection process to change original length of tested fragment, reaches tested fragment in identification spatially
In an orderly manner misplace and expand fragment length detection range, thus can not only extend single fluorescent dye can labelling locus
STR segments, and can solve the problem that pleochroic dye (more than 5 colors) in labelling STR fragment because more serious fluorescence spectrum is intersected
Problem and lead to instrument detection when be difficult to determine each dye marker fragment gene seat ownership (gene type) problem.
The mentality of designing of the present invention is as shown in Figure 1.
The inventive method includes two parts, is respectively directed to two kinds of different purposes：
1. expand the STR segment number that single fluorescent dye is capable of labelling.
The method illustrating in Fig. 2 to expand single fluorochrome label STR segment number, as shown in Figure 2 a, first, individually
The Single-stranded DNA fragments that amplification obtains a length specific (for example, 220bp) are replicated by chemosynthesis and biosynthesiss round pcr,
The Single-stranded DNA fragments upper band of these synthesis has the functional group for being connected chemically；Then, when designing primer, to fluorescent labeling
Forward primer make the design that is slightly changed in a chemical constitution, allow the primer under some locus keep normal dyestuff mark
Note structure, and the primer under other locus first introduced one before dye marker and carries the foot handss that can be used for being connected chemically
Frame molecule；At least can occur containing three on each scaffold molecule being introduced in three functional groups of functional group of chemical reaction
One functional group is connected with primer, and a functional group is connected with dyestuff, and also a functional group is connected with Single-stranded DNA fragments, this
Sample can make the 5 ' of primer to hold with upper one Single-stranded DNA fragments being connected chemically with scaffold molecule；This single stranded DNA
The length of fragment can require to adjust and change according to different detections；As shown in figs. 2 b and 2 c, performing PCR is being entered to STR fragment
After replicating or expanding, the pre-synthesis specific Single-stranded DNA fragments of length are added in the solution of PCR purified product, quilt
The STR fragment being connected with the primer amplification of scaffold molecule then passes through foot with the pre-synthesis specific Single-stranded DNA fragments of length
Respective functional group on hand cradle molecule and Single-stranded DNA fragments is connected chemically reaction, and the response time is generally 15-20 and divides
Clock, after completion of the reaction solution system can produce the fragment of two kinds of different lengths, a kind of is not to be connected chemically reaction
Former PCR fragment (fragment 1), its length is not changed in；Another kind is to have occurred and that being connected chemically reaction leads to the new of length growth
Fragment (fragment 2), the sample containing fragment 1 and the mixed solution of fragment 2 can be detected by normal Instrumental Analysis, originally
Length identical fragment, because a length increases, occurs the separation in detection time accurately to be detected in electrophoresis.
Using this method, the locus number that each dyestuff is capable of labelling can increase about one times, thus can will be various at present
Limited locus number in STR detection extends about one times；For example, used in current identity authentication, 21 sites just extend to
More than 40 sites.
The primer of the dye marker of introducing scaffold molecule used in the inventive method in structure can be in dyestuff, foot
Hand cradle molecule, is provided with spacer molecule between primer three, in same fragment, spacer molecule can be identical it is also possible to
It is different, used spacer molecule can be linear or bifurcated, such as linear structure is NH2-(CH2)n-
COOH (n=1,2,3,4 ... .16), spacer molecule and dyestuff, scaffold molecule, the connection of primer is as shown in Figure 6.
Separately through chemistry and biosynthesiss round pcr synthesize a segment length specific and 5 ' end band one can be with scaffold
The sequence of the Single-stranded DNA fragments that the functional group of molecule is connected chemically and the layout strategy of length：
Above-mentioned Single-stranded DNA fragments sequence can use arbitrary DNA sequence, but must follow following former during selection sequence
Then：
(1) sequence has enough stability, including difficult to form hairpin structure, and avoids restriction enzyme site etc.；
(2) it is readily synthesized；
The length of Single-stranded DNA fragments mainly according to depending on traditional detection sequence length range it is however generally that, this piece
The sequence length of section reaches or just above traditional detection range, for example, when traditional detection sequence length range is
During 80-350bp, the fragment length of design can use the span of this scope, i.e. 270bp (350-80=270).
The scaffold molecule introducing is molecule with chemical reaction functional group, typically by a basic framework and available
In the group composition being connected chemically.
Skeleton may include following three kinds：
A. with D-, or L-, or the scaffold that DL-Lys or aminoacid that other similar side chains are amino are skeleton divides
Son, shown in framing structure such as formula (1) or (2)：
Wherein, n=1 in formula (1) and (2), 2,3,4, R is the functional group for being connected with Single-stranded DNA fragments, on skeleton
Amino can be included within R functional group；
B. with D-, or L-, or DL- winter propylhomoserin, glutamic acid or the aminoacid that other similar side chains are carboxyl are skeleton
Scaffold molecule, shown in framing structure such as formula (3)：
Wherein, in formula (3), n=1,2,3, R is that on skeleton, carboxyl is permissible for the functional group being connected with Single-stranded DNA fragments
It is included within R functional group；
C. with D-, or L-, or the scaffold that DL-cysteine or aminoacid that other similar side chains are sulfydryl are skeleton
Molecule, shown in framing structure such as formula (4)：
Wherein, in formula (4), n=1,2；
The scaffold molecule of other chemical functional groups, bone, d. with nucleotide units as basic framework, are introduced on its base
Shown in frame structure such as formula (5)：
The group being used on skeleton being connected chemically has the structure shown in formula (6-14)：
Formula (6) and (7) are maleimide, and in formula (7), R is straight chained alkyl；
Formula (8) is iodoacteyl；
Formula (9) is sulfydryl；
When the group connecting is amino, including chemical conversioning reaction can be passed through, such as with Traut reagent reacting, with N-
Acetyl group-homocysteinic acid thiolactone reaction conversions become the amino of sulfydryl；
Formula (10) is azido；
Formula (11) is alkynyl；
Formula (12) is diazanyl, and formula (13) is azyloxy
In formula (14), R1=H or straight chained alkyl, R2For straight chained alkyl or alkoxyl；
Functional group on Single-stranded DNA fragments can be for having formula (6) structure shown in (14).
Embodiment 1
In DNATyper 15 Plus identity authentication test kit, with 12 locus of dyestuff FAM labelling
(Amelogenin*2, D5S818*2, D21S11*2, D7S820*2, CSF1P0*2, D3S1358*2), in dyestuff FAM and primer
A scaffold molecule being connected to there is structure functional group shown in formula (8) is added between DNA fragmentation；After the completion of PCR, will be pre-
It is molten that one section of Single-stranded DNA fragments of first synthetic length specific (300bp) are added to the mixing obtaining after PCR amplification purification
In liquid, it is connected chemically reaction after 20 minutes and completes；The solution of gained carries out capillary electrophoresis and fluoroscopic examination；Newly-increased locus
Although the dyestuff of labelling used is identical, the piece size of labelling is different, there occurs certain distance on identification spatial arrangement
Displacement, completely not overlapping with the size range of original detection, therefore these newly-increased locus can be detected and differentiate, from
And realize gene type.
2., solve the more serious fluorescence spectrum cross-cutting issue of the pleochroic dye of labelling STR fragment taking four color dyestuffs as a example.
As Fig. 3, shown in 4,5, in design of primers, fluorescence spectrum is intersected serious dyestuff (D1, D2, D3, D4, such as Fig. 3
Shown) first it is divided into two groups of (D1And D3For one group, D2And D4For another group, allow every group dye fluorescence spectrum intersection as far as possible
Little), in labeled primer, conventional labels method is adopted to the dyestuff of one of which, and another group adopts the single fluorescent dye of expansion
The method of labelling STR segment number, first introduces a scaffold molecule between dyestuff and DNA primer fragment；To STR piece
After section enters performing PCR amplification, the pre-synthesis specific Single-stranded DNA fragments of length are added to the mixing of this PCR purified product
In solution, be connected with scaffold molecule primer amplification STR fragment then with pre-synthesis length specific single stranded DNA piece
Section is connected chemically reaction, reaction to be connected chemically by the respective functional group on scaffold molecule and Single-stranded DNA fragments
After end, comprise in this mixed solution to keep the constant fragment 1 of length and fragment 3, fragment 2 and piece that also length has increased
Section 4 (as shown in Figure 4)；When mixed solution being tested and analyzed by instrument, because part STR fragment is connected to a length
Specific Single-stranded DNA fragments, hold, lead to time of staying during electrophoresis delayed, therefore avoid the dyestuff mark that spectral cross is occurred
The situation that the fragment of note is simultaneously detected, thus avoid more serious fluorescence cross problem between multiple dyestuffs.
Four primers by dye marker, in figure D is shown in Fig. 31, D2, D3, D4For dyestuff, D1And D3The primer of labelling
Structure is identical, D2And D4The primer of labelling adds a scaffold molecule between dyestuff and primer.Fig. 4 represents 4 in Fig. 3
Primer is used for STR fragment, and after PCR amplification, specific Single-stranded DNA fragments are connected chemically with length again, only dyestuff D2And D4
The fragment 2 of labelling and fragment 4 just can occur fragment to connect, and length increases, and dyestuff D1And D3The fragment 1 of labelling and fragment 3 because
Can not be connected chemically, length is constant.
Fig. 5 represents that four fragments occur in electrophoresis to separate, wherein dyestuff D1And D3The fragment 1 of labelling and fragment 3 are because of length
Identical, electrophoresis translational speed phase is in one group together, and dyestuff D2And D4There is position in the fragment 2 of labelling and fragment 4 on spatial arrangement
Move, with dyestuff D1And D3The fragment 3 of fragment 1 sum of labelling separates and is in another group, such D1And D2、D2And D3、D3And D4To each other
Because of fragment 1, fragment 2, fragment 3, fragment 4 difference is no longer detected fluorescence spectrum at the same time, thus avoiding fluorescence spectrum to intersect
Interference.
Embodiment 2
In the identity authentication of eight 40 locus of color fluorochrome label, eight color dyestuffs can be FAM, HEX, TMR,
ROX,Cy5,Cy5.5,Cy7,Cy7.5；Due to intersecting due to fluorescence spectrum, eight color dyestuffs can be divided into two groups, A group is FAM,
TMR,Cy5,Cy7；B group is HEX, ROX, Cy5.5, Cy7.5；For the primer of A group echo, using conventional labeling method；And
For B group dye marker primer then using the method expanding single fluorochrome label STR segment number, that is, in dyestuff and
A scaffold molecule is first introduced between DNA primer fragment；After STR fragment is entered with performing PCR amplification, by pre-synthesis length
Spend in the mixed solution that specific Single-stranded DNA fragments are added to this PCR purified product, the primer being connected with scaffold molecule expands
The STR fragment increasing then is passed through on scaffold molecule and Single-stranded DNA fragments with the pre-synthesis specific Single-stranded DNA fragments of length
Respective functional group is connected chemically reaction, is connected chemically reaction and completes after 20 minutes.The solution of gained carries out capillary tube electricity
Swimming and fluoroscopic examination, produce the DNA piece all with a length-specific for the dye marker DNA fragmentation that fluorescence spectrum intersects due to easy
Duan Fasheng is connected chemically thus leading to fragment to increase, and has displacement with the fragment originally having spectral cross, no longer on spatial arrangement
It is detected simultaneously, thus each fragment all can be easier to detected and differentiate, and reach the purpose of gene type.
Finally it should be noted that：Various embodiments above only in order to technical scheme to be described, is not intended to limit；To the greatest extent
Pipe has been described in detail to the present invention with reference to foregoing embodiments, it will be understood by those within the art that：Its according to
So the technical scheme described in foregoing embodiments can be modified, or wherein some or all of technical characteristic is entered
Row equivalent；And these modifications or replacement, do not make the essence of appropriate technical solution depart from various embodiments of the present invention technology
The scope of scheme.
Claims (10)
1. a kind of limited loci STR analysis method it is characterised in that：The method is included using by the primer of fluorochrome label
Limited loci STR fragment is entered performing PCR replicate and amplification after specific for length Single-stranded DNA fragments can be connected to portion
Divide in the limited loci STR fragment after amplification.
2. limited loci STR analysis method according to claim 1 it is characterised in that：Described by fluorochrome label
Primer is forward primer or reverse primer in two-way primer, and before by fluorochrome label, draws in partly described forward direction
One scaffold molecule is introduced on thing or partly described reverse primer.
3. limited loci STR analysis method according to claim 2 it is characterised in that：Described scaffold molecule includes base
This skeleton and the functional group being connected chemically with described Single-stranded DNA fragments on described basic framework.
4. limited loci STR analysis method according to claim 3 it is characterised in that：Described basic framework includes having
Structure shown in formula (1) or (2),
In formula (1) and (2), n=1,2,3,4, R is the functional group on described skeleton；
Or there is the structure shown in formula (3),
In formula (3), n=1,2,3, R is the functional group on described skeleton；
Or there is the structure shown in formula (4),
In formula (4), n=1,2；
Or there is the structure shown in formula (5),
5. limited loci STR analysis method according to claim 2 it is characterised in that：Described Single-stranded DNA fragments are to pass through
The primer of chemosynthesis and biosynthesiss PCR replicate the specific fragment of a segment length that amplification obtains, and its sequence is usual and tested
Sample is unrelated, and its length and sequence can adjust according to detection requirement and change；Have on described Single-stranded DNA fragments can with described
The functional group that scaffold molecule is connected chemically.
6. the limited loci STR analysis method according to any one of claim 3 to 5 it is characterised in that：Described functional group is
In maleimide, haloacetyl, amino, sulfydryl, azido, alkynyl, diazanyl, azyloxy, aldehyde radical or ketone group at least one
Kind.
7. limited loci STR analysis method according to claim 6 it is characterised in that：Described amino is also included by changing
Learn the sulfydryl that conversion reaction is changed generation further by amino；Described chemical conversioning reaction include with Traut reagent or with N- second
The chemical reaction that acyl group-homocysteinic acid thiolactone reagent occurs.
8. limited loci STR analysis method according to claim 6 it is characterised in that：Described maleimide have as
Structure shown in formula (6) or formula (7),
In formula (7), R is straight chained alkyl；Described aldehyde radical or ketone group have the structure as shown in formula (14),
In formula (14), R1=H or straight chained alkyl, R2For straight chained alkyl or alkoxyl.
9. limited loci STR analysis method according to claim 2 it is characterised in that：Described fluorescent dye, scaffold divide
Sub with described forward primer or described fluorescent dye, scaffold molecule are provided with spacer molecule between any two with described reverse primer；
In same locus STR fragment, the structure of described spacer molecule can be identical or different, the structure bag of described spacer molecule
Include linear structure or bifurcation structure.
10. a kind of DNA fragmentation connects displacement dilatation and goes application in limited loci STR analysis for the spectrum cross-cut analysis method.
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| CN110265088A (en) * | 2019-06-14 | 2019-09-20 | 孙翊鸣 | A kind of method that DNA is converted into pattern |
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| CN102703595A (en) * | 2012-06-13 | 2012-10-03 | 东南大学 | STR (short tandem repeat) sequence high-throughput detection method with base selective controllable extension and detection reagent thereof |
| CN103917661A (en) * | 2011-05-12 | 2014-07-09 | 网络百奥有限公司 | Methods and compositions for rapid multiplex amplification of STR loci |
| CN104152568A (en) * | 2014-08-19 | 2014-11-19 | 东南大学 | High-flux STR sequence core replication number detection method |
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| CN103917661A (en) * | 2011-05-12 | 2014-07-09 | 网络百奥有限公司 | Methods and compositions for rapid multiplex amplification of STR loci |
| CN102703595A (en) * | 2012-06-13 | 2012-10-03 | 东南大学 | STR (short tandem repeat) sequence high-throughput detection method with base selective controllable extension and detection reagent thereof |
| CN104152568A (en) * | 2014-08-19 | 2014-11-19 | 东南大学 | High-flux STR sequence core replication number detection method |
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| CN110265088A (en) * | 2019-06-14 | 2019-09-20 | 孙翊鸣 | A kind of method that DNA is converted into pattern |
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