阅读说明：本技术 用于通过使用集成电泳dna纯化来检测遗传结构变异的系统和方法 (For purifying the system and method to detect genetic structure variation by using integrated electrophoresis DNA ) 是由 T·C·伯勒斯 于 2018-04-06 设计创作，主要内容包括：一种电泳盒,其可以包括样品孔、含有分离凝胶的凝胶柱和与凝胶柱相邻布置的洗脱模块。样品可以被提供给电泳盒,并且可以从样品中分离高分子量DNA。可以在DNA序列的重复区域的两侧上切割单拷贝DNA序列,以产生切割的样品,然后可以使用凝胶电泳对其进行分级。可以从分离凝胶的连续切片中分离DNA级分,并且经受PCR测定,以检测DNA级分内的单拷贝序列,所述单拷贝序列含有重复扩增序列。可以将所述DNA级分电洗脱到多个洗脱模块内。可以确定具有重复扩增序列的DNA级分的大小。还确定该大小是否在正常重复大小范围以上。(A kind of electrophoresis cartridge may include sample well, the gel column containing separating gel and the elution module being adjacently positioned with gel column.Sample can be provided to electrophoresis cartridge, and high-molecular-weight DNA can be separated from sample.Single-copy DNA sequence can be cut on the two sides of the repeat region of DNA sequence dna, to generate the sample of cutting, gel electrophoresis then can be used, it is classified.DNA fraction can be separated from the serial section of separating gel, and is subjected to PCR measurement, and to detect the single-copy sequence in DNA fraction, the single-copy sequence contains repeat amplification protcol sequence.It can will be in the DNA fraction electroelution to multiple elution modules.It can determine the size of the DNA fraction with repeat amplification protcol sequence.Also determine whether the size is normally repeating magnitude range or more.)

1. a kind of method comprising:

There is provided includes electrophoresis cartridge below:

At least one sample well,

At least one gel column containing separating gel, and

The multiple elution modules being adjacently positioned at least one described gel column；

Sample is provided to the electrophoresis cartridge；

High-molecular-weight DNA is separated from the sample；

Single-copy DNA sequence is cut, on the two sides of the repeat region of the DNA sequence dna to generate the sample of cutting；

The sample of the cutting is classified using gel electrophoresis；

DNA fraction is separated from the serial section of the separating gel；

The DNA fraction is set to be subjected to PCR measurement, to detect the single-copy sequence in the DNA fraction, the single-copy sequence contains There is repeat amplification protcol sequence；

It will be in the DNA fraction electroelution to the multiple elution module；

Determine the size with the DNA fraction of the repeat amplification protcol sequence；And

Determine whether the size of the DNA fraction with the repeat amplification protcol sequence is normally repeating magnitude range or more.

2. according to the method described in claim 1, wherein described cut through restriction enzyme execution.

3. according to the method described in claim 2, wherein the restriction enzyme is configured to do not containing duplicate DNA fragmentation inscribe It cuts.

4. according to the method described in claim 1, wherein the cutting is executed with the nickase that customized RNA or DNA is instructed.

5. according to the method described in claim 4, wherein the nickase of the RNA or DNA guidance is one of following or more Kind: Cas9, Cpf1 and NgAgo.

6. according to the method described in claim 1, its further include:

In the multiple elution module provide liquid electrophoretic buffer so that electroelution to it is the multiple elute module in institute DNA fraction is stated to be arranged in the electrophoretic buffer；

It will be added in PCR reaction with the electrophoretic buffer of the DNA fraction；And

The single-copy sequence target of the DNA fraction of the PCR reaction is measured in the repeat amplification protcol sequence.

7. according to the method described in claim 1, the condition for wherein changing the electrophoresis changes the movement of the DNA fraction Property.

8. according to the method described in claim 7, wherein the deposition condition includes one of following or a variety of: gel is dense Degree, voltage, voltage waveform, buffer composition and runing time.

9. according to the method described in claim 7, wherein the deposition condition is changed in such a way that the DNA fragmentation on predetermined length Slow down and is moved at least one gel column from far from electrophoresis.

10. a kind of system comprising:

For separating the electrophoresis cartridge of high-molecular-weight DNA from sample, the electrophoresis cartridge includes:

At least one sample well,

At least one gel column containing separating gel, and

The multiple elution modules being adjacently positioned at least one described gel column；

Cutting reagent, to cut the single-copy DNA sequence on the two sides of the DNA repeat region, to generate the sample of cutting Product；With

It is configured to detect the PCR measurement of the single-copy sequence in the DNA fraction.

Background technique

Many heredity genetic diseases are caused by the length amplification containing the duplicate chromosomal region of simple DNA sequence dna.Example Such as, fragile X mental retardation syndrome, by (CGG) near the end 5' of gene FMR1_nSequence causes, and from most In the unaffected individual of number < more than 200 copies of 50 CGG copy amplifications into most of impacted individuals (Nolin et al., 2003).Similarly, in the most common mutated gene relevant to ALS, C9orf72, First Intron In (G₄C₂)_nRepetition repeats to>=300 duplicate amplifications (Suh et al., 2015) related to morbid state from<8.At least 22 kinds of hereditary neurological diseases cause (La Spada and Taylor, 2010) by the mutation of this class repeat amplification protcol.

The detection and analysis of such repeat amplification protcol mutation may be complicated because of several factors.Firstly, being 2- containing length The PCR amplification in the region of the simple sequence repeats of 10bp be it is error-prone, generally produce different in terms of number of repeat unit Amplicon product family.Many repeat amplification protcols are also extremely rich in GC, this makes PCR measurement exploitation even more difficult.It is logical The careful optimization for specific gene group locus is crossed, these problems can be preferably minimized, and allow to obtain useful diagnosis Measurement, but the optimization of such measurement is laborious and time-consuming, and the condition about a kind of repeat amplification protcol type generally can not It is transferred to other measurements.

About PCR measurement another difficulty is that the size of some repeat amplification protcols can with > 20kb (Nolin et al., 2003), The typical size range for having exceeded PCR measurement, is generally found that between 5-10kb.This means that having the equipotential greatly expanded Gene may be not detected in PCR measurement.

In order to avoid these complications, still analyzed in many cases using southern blotting technique, it is especially big in repeat amplification protcol It is small can be many kb in the case where.However, southern blotting technique is conventional using being very laborious and time-consuming, and result out Time can be two to four days, including the engram analysis time.

Summary of the invention

This document describes various instruments, system and method.In some embodiments, electrophoresis cartridge can be provided.Electrophoresis cartridge can To include at least one sample well, at least one gel column containing separating gel and be arranged against at least one gel column Multiple elution modules.Sample can provide in electrophoresis cartridge.It can be from wherein separating high-molecular-weight DNA, and it can be in DNA Repeat region two sides on cut single-copy DNA sequence, to generate the sample of cutting.Gel electrophoresis classification can be used to cut The sample cut, and DNA fraction can be separated from the serial section of separating gel.DNA fraction can be made to be subjected to PCR measurement, To detect the single-copy sequence in DNA fraction, the single-copy sequence contains repeat amplification protcol sequence, and can be by the DNA In fraction electroelution to multiple elution modules.It can determine the size of the DNA fraction with repeat amplification protcol sequence.It can determine tool Have whether the size of the DNA fraction of repeat amplification protcol sequence is normally repeating magnitude range or more.

Cutting can be executed by restriction enzyme, and these enzymes are configurable to not containing in duplicate DNA fragmentation Cutting.Alternatively and/or in addition, cutting, the cutting can be executed with the nickase that customized RNA or DNA is instructed Enzyme may include Cas9, Cpf1 and NgAgo.

In some embodiments, liquid electrophoretic buffer can be provided in multiple elution modules of electrophoresis cartridge, so that through In the DNA fraction electroelution to multiple elution modules measured by PCR, and it is arranged in the electrophoretic buffer.Can will have The electrophoretic buffer of DNA fraction is added in PCR reaction, and this can be carried out with regard to the single-copy sequence target in repeat amplification protcol sequence Measurement.

Change deposition condition, such as gel strength, voltage, voltage waveform, buffer composition and runing time, thus it is possible to vary The mobility of DNA fraction.Can change condition so that the DNA fragmentation on predetermined length slow down from far from electrophoresis be moved to In a few gel column.

Detailed description of the invention

Figure 1A shows the amplification of sequence repeat region in accordance with some embodiments.

Figure 1B shows the insertion of sequence in accordance with some embodiments.

Fig. 1 C shows the reversion of sequence in accordance with some embodiments.

Fig. 2A -2B shows the length in accordance with some embodiments for not expanding repetition and single copy flanking sequence.

Fig. 2 C shows that amplification in accordance with some embodiments repeats the length with single copy flanking sequence.

Fig. 3 shows exemplary process diagram in accordance with some embodiments.

Fig. 4 A-4C shows the position of single copy qPCR detection amplicon in accordance with some embodiments.

Fig. 5 A is shown according to some embodiments, is then the SageELF box of electroelution for DNA size separation.

Fig. 5 B shows according to some embodiments, from separation electrophoresis to classification/the exemplary SageELF work of electroelution Stream.

Fig. 6 shows exemplary SageHLS box in accordance with some embodiments.

Fig. 7 A-10B shows exemplary workflow according to various embodiments.

Figure 11 A-11B shows the deposition condition according to some embodiments, on SageHLS box.

Figure 12 shows illustrative diagram in accordance with some embodiments.

Figure 13 A-13B shows the figure for the result realized in example 1 according to some embodiments.

Specific embodiment

Disclosed herein is the program for characterizing repeat amplification protcol mutation, the wide dimensional flexibility southern blotting technique measurement of a combination thereof With the detection by PCR.Many measurements are applied, workflow can be completed in less than one day.

Figure 1A shows the genomic DNA before amplification 105, with single-copy sequence A110, contains simple sequence repeats 115 chromosomal region and single-copy sequence B120.Genomic DNA before amplification 105 has length 125.When amplification contains letter When simple sequence repeats 115 chromosomal region, disease condition 130 can produce.The simple sequence repeats region 130 of amplification is one Side is surrounded by single-copy sequence A110', and is surrounded in other side coverlet copy sequence B 120'.The length of the A-B segment 125' of amplification Degree is longer than the length of A-B segment 125.

As it is shown in the figures, ' > ' indicates simple sequence repetition unit.For example, this can be in als gene C9orf72 G₄C₂.In C9orf72, G relevant to disease phenotype₄C₂The threshold estimation of repetition number is between 30 and 70, although many Impacted individual can have the repeat amplification protcol for arriving tens of thousands of a bases (thousands of recurring units) greatly.

Figure 1B shows the insertion of sequence in accordance with some embodiments.Herein, the genomic DNA 135 before insertion has single Copy sequence A140, chromosomal target site 145 and single-copy sequence B150 for insertion event.A-B segment has length 155.Then sequence is inserted at target site 160.After such insertion, single-copy sequence A140' is located at the side of insetion sequence 160 On, and single-copy sequence B150' is located on the other side.The length of obtained A-B segment 155' is longer than A-B segment after insertion 155 length.

Fig. 1 C shows the reversion of sequence in accordance with some embodiments.As indicated, genomic DNA 165 has single copy sequence Arrange A170 and single-copy sequence B175.Single-copy sequence A170 can be only fitted to left end and the reversion breakpoint 185 of reversion breakpoint 180 Right end between.Single-copy sequence B175 can be only fitted to except reversion breakpoint 180,185.A-B segment can have length 190.The section inverted between the left end of breakpoint 180 and the right end of reversion breakpoint 185 can be inverted 195.Obtained gene Group DNA 165' can have the single-copy sequence A170' and single-copy sequence B175' being configured so that, so that the A-B after reversion The length of segment 190' is different from the length of A-B segment 190.In some embodiments, such as shown in Fig. 1 C that, reversion The length of A-B segment 190' afterwards can be longer than the length of the A-B segment 190 before reversion.In some embodiments, after reversion The length of A-B segment 190' can be shorter than the length of the A-B segment 190 before inverting.

Fig. 2A shows base before being cut with specific cleavage site A 205 and specific cleavage site B 210 Because of group DNA 200.Containing the configuration of duplicate chromosomal region 215 between cleavage site A205 and cleavage site B 210.Display The length 220 of repetition and single copy flanking sequence is not expanded.In fig. 2b, genomic DNA 200 is in cleavage site A 205 It is cut at cleavage site B 210, so that cutting section has length 220.Fig. 2 C, which is shown, has amplification repeat region 225 Specific cleavage site A 205' and specific cleavage site B 210'.The repetitive sequence of amplification and single copy flanking sequence tool There is length 220 '.

Exemplary embodiment is shown in Fig. 3.Sample 305 can be provided, and HMW DNA can be separated from sample 310.Specificity cutting 315 can be executed with single-copy sequence on the two sides of the repeat region of HMW DNA.The sample of cutting can To be classified.In some embodiments, using the sample of gel electrophoresis classification cutting.The selection of electrophoresis size can be used effectively Ground, which is differentiated to carry not expanding duplicate genomic fragment and carry, expands duplicate segment to execute 320.It can be from separating gel And/or DNA 325 is separated in continuous/abutting sections of gel lane.DNA fraction can be subjected to PCR 330.PCR can be pass In flank repetitive sequence, in the single-copy sequence in the repeat region of excision amplicon measurement.It can determine from elution Position in fraction is the DNA size 335 in positive elutriated fraction for the scoring of repeat amplification protcol region, and can determine and appoint What amplicon whether in being greater than the normal genomic fragment for repeating magnitude range detect 340.

In some embodiments, the basis of measurement is the single copy of uniqueness measured by the two sides in repeat amplification protcol region The length (Figure 1A, Fig. 2A-B) for the DNA fragmentation that cutting at DNA sequence dna generates.From do not expand duplicate segment be less than be originated from Expand duplicate segment.Size fractionation carried out to the sample of cutting by gel electrophoresis, and from the serial section of separating gel Middle separation DNA, including all gel areas occupied by sample DNA.The DNA fraction of purifying is set to be subjected to PCR measurement, the PCR Measurement is designed as detecting the single-copy sequence (Fig. 4 A-4C) in the specificity release segment containing repeat amplification protcol sequence.

As shown in Figure 4 A, it is shown that the gene with specific cleavage site A 405 and specific cleavage site B 410 Group DNA 400.The configuration of chromosomal region 140 containing repetitive sequence is in specific cleavage site A 405 and specific cleavage Between point B 410.Fig. 4 B shows the cutting at specific cleavage site A 405 and specific cleavage site B 410.In spy Anisotropic cleavage site A 405 and contain be between duplicate chromosomal region 415 single copy qPCR detection amplicon 420 position It sets.Fig. 4 C shows the amplification repeat region 425 between specific cleavage site A 405' and specific cleavage site B 410'. Show the position of single copy qPCR detection amplicon 420'.

Limitation can be passed through in the cutting (including flank single-copy sequence (Fig. 2A -2C)) that disclosure discusses from beginning to end Property enzyme realize that condition is them not containing other place cuttings in duplicate segment.These cuttings can also be with can determine Nickase such as Cas9, Cpf1 or NgAgo of RNA or the DNA guidance of system are completed.

In some embodiments, the genomic DNA fragment of digestion is in Fig. 5 A, Fig. 5 B (also in U.S. Patent number 9,599,590 Description in (it is hereby incorporated herein by)) or Fig. 6 (also in PCT Application No. PCT/US2015/055833, (it is with reference Mode be incorporated herein) in description) shown in size separation and electroelution are carried out in electrophoresis cartridge.After size fractionation, it will divide In entire DNA content transverse direction electroelution to the elution module for the adjoining series being arranged on separating gel column side from gel. By the DNA electroelution of classification to elution module in liquid electrophoretic buffer in, and can be directly added into PCR reaction in, and Measure the single-copy sequence target in the repeat amplification protcol segment of excision.Since the DNA size in each elutriated fraction is by deposition condition (such as gel percentage, buffer, runing time, voltage) determines, therefore the position of positive PCR signal can be in elutriated fraction It is directly related with the size of the repeat region allele detected.

In some embodiments, instrument, method and system described in PCT/US2015/055833 are for completing to own Step before PCR.Exemplary workflow is shown in schematic form in Fig. 7 A-10B.Such as PCT Application No. PCT/US2015/055833 Described in, high molecular weight genomic DNA can be extracted and digested in an integrated workflow, with the user of bottom line Intervene.Generate the DNA restriction enzyme that the specificity cutting of detectable repeat amplification protcol segment can use DNA restriction enzyme for example traditional The nickase such as micrococcus scarlatinae (Streptococcus pyogenes) of (Fig. 7 A-7E and Fig. 8 A-8B) or RNA guidance Cas9 (Fig. 9 A-9E and Figure 10 A-10B) is completed.It can be used input material, such as the white blood corpuscle, unassorted complete of purifying Blood and the cell suspending liquid obtained from the tissue sample of dissociation (or core).

As shown in Figures 1 B and 1 C, system and method disclosed herein can also be used for detection insertion and reversion is reset.In In the case of two kinds, reset change flank rearrangement a breaking point (i.e. insertion point, Figure 1B, or reversion a breaking point, Fig. 1 C) the distance between unique cleavage site.

As mentioned in the Introduction, in some repeat amplification protcol diseases, amplification can considerably long and alterable height.In order to solve this A problem can modify deposition condition (including such as gel strength, voltage, voltage waveform, buffer composition, runing time), So that all DNA moleculars for being greater than certain length are all migrated as restricted low mobility fraction together.This occurs by length Increase (that is, the charge from phosphate backbone increases) in caused electrophoretic mobility is drawn by the resistance increase of bigger molecule When the reduction in electrophoretic mobility risen is offset.Molecular size at the restricted low mobility point be gel percentage, The compound function of voltage and buffer composition.However, for given buffer and gel strength, about the restricted low of DNA Mobility can be adjusted in the range of 1000bp is until multiple 10,000bp in Ago-Gel.Figure 11 A-11B is aobvious The deposition condition (as described in PCT/US2015/055833) on SageHLS box is shown, wherein can compile in elution module The restricted low mobility band started with 10,000bp is eluted in numbers 2.

In some embodiments, the deposition condition about specific repeat amplification protcol locus can be modified, so that do not expand Repeated fragment elutes near the bottom of gel column, and the repeated fragment moderately expanded is not expanding in the intermediate range of elutriated fraction Increase and differentiated in the fraction on fraction, and has the segment that greatly expands in the restricted low migration of the near top of gel column (Fig. 8 A-8B and Figure 10 A-10B) is eluted in rate compression strap.

Example 1. is for the integrated extraction of specific chromosomal loci, Cas9 digestion, electroelution and qPCR measurement The demonstration of SageHLS workflow

This example illustrate SageHLS purification of high molecular weight genomic DNA from input cell sample is used, Cas9 is used Nickase selectively cuts off specific 198kb genomic DNA fragment from BRCA1 locus, and finally, integrated at one Workflow in size selection and elution the segment containing BRCA1.Then the BRCA1 piece of HLS elutriated fraction is measured by pPCR Section.

Buffer definition:

Electrophoretic buffer, also referred to as 0.5xKBB (51mM Tris (alkali), 29mM TAPS (acid), 0.1mM EDTA (acid), pH 8.7)

FSE buffer: 15%w/v Ficoll 400,0.25xKBB buffer, 80mg/mL sucrose, 10mM EDTA

ERB buffer: 0.5xKBB, with 32mg/ml beta-cyclodextrin, 10mM MgCl₂, 50 μ g/ml BSA addition

HLS lysis buffer: 1xKBB, 2% glycerol, 3%SDS, 2.5 μ g/ml bromophenol blues, 2.5 μ g/ml are phenol red

People is cultivated into cell (Raji cell line) washing for several times by low-speed centrifugal, and is resuspended to phosphate buffer salt In water.After last time is washed, by cell with every 70 microlitres of 1.5X10⁶The concentration of a cell is resuspended in FSE buffer.In Two 70 microlitres of samples of the cell of resuspension are loaded into two sample wells of SageHLS box (0.75% agarose) in FSE Each in.The reagent wells of two swimming lanes are emptied, and are refilled with HLS lysis buffer (about 230 microlitres), and And it is carried out electrophoresis 1 hour at 30 DEG C, 55V.

After purifying electrophoresis, sample well and reagent wells are emptied.Reagent wells are refilled with ERB buffer (being free of enzyme).In In one of two swimming lanes, sample well 80ul contains 1 micromole's wt micrococcus scarlatinae Cas9 enzyme (New England Biolabs ERB) is refilled, and the enzyme has used the equimolar mixture of 5 two parts guide RNAs to assemble, and respectively 5 is micro- Molar concentration.In another swimming lane, the ERB without enzyme is loaded into sample well as simulation digestion control.By HLS instrument Sample well heater is adjusted to 37 DEG C, and electrophoresis is moved in gel 1 minute at 55V by Cas9 mixture.In 1 minute electricity After swimming, sample well is emptied and is refilled with the ERB buffer without enzyme.Box is incubated 30 in the case where no electrophoresis Minute, wherein sample well incubates at 37 DEG C, to allow to purify the Cas9 digestion of DNA.

After digestion, by reagent wells empty and refilled with HLS lysis buffer, and use be designed as by 200kb BRCA1 digestion product move to elution module 34 hours pulsed field programs carry out size separation electrophoresis (HLS-CATCH's 3 program 100-400kb, SageHLS user's manual of stage, Sage Science, Inc.).After size separation, use 50V's Continuous field voltage carries out electroelution 1.5 hours.

Two parts guide RNA (ALT-R is ordered from IDT^TMCrRNA and tracRNA).Select gRNA to cut off 198kb segment, It includes entire BRCA1 locus, has sufficient flanking sequence on the side 5' and 3' (referring to Figure 12).It devises for gene Right side three kinds of crRNA--BRCA1gR67:GCTTATTACATTCTCGGCCA；BRCA1gR68: CTTATTACATTCTCGGCCAT；And BRCA1gR69:ATTACATTCTCGGCCATGGG.It devises for for gene Two kinds of crRNA--BRCA1gLL1:CCTCTGGGAGCCACAGGCCA in left side；And BRCA1gLL3: GCCATGACAACAACCCAGAC (Figure 12).CrRNA and tracRNA (IDT) are dissolved in IDT double buffering liquid, and led to Crossing will exist containing the mixture of 50 micromole tracRNA and each 10 micromolar 5 kinds of crRNA (50 micromole in total in crRNA) It is incubated 5 minutes at 95 DEG C, and cooling 15 minutes on table top under ambient lab temperature anneal.Be added HLS box it Before, by the assembling in ERB buffer (seeing above) by final reacting mixture, and mixture is incubated 10 points at 37 DEG C Clock assembles gRNA the and Cas9 enzyme of annealing.

After elution, by the product of elution, 1:10 dilutes in 10mM Tris-HCl, 1mM EDTA, pH 8.0, and leads to Taqman qPCR measurement BRCA1 gene DNA is crossed, uses RNaseP rna gene as the reference locus about non-target DNA. (ABI/Life Technologies unit number: the measurement of #4400291-BRCA1 copy number (Hs00300666-cn amplicon, It is small)；#4403326-RNaseP copy number is with reference to measurement；#4371355-Taqman GT Master Mix；QPCR instrument；ABI QuantStudio 3).BRCA1 has been recycled in the fraction 3 of the box swimming lane of Cas9 digestion as the result is shown in Figure 13 A-13B The 1.5x10 of segment⁶The recycling of a copy, but the only visible background signal in the box swimming lane of simulation digestion.

Example 2. can be used for detecting the demonstration of the gel compression of macromolecular structure variant.

The sample of DNA marker (1kb extends marker, New England Biolabs) is loaded into SageHLS box In the sample well of two swimming lanes.By DNA separation and following deposition condition electroelutions: 0.75% agarose are used, 50mM Tris, 29mM TAPS, 0.1mM EDTA, pH 8.7, the continuous field 55V (DC), 50 minutes, 30 DEG C of gelling temp.It is flat in analysis agarose The electroelution fraction (Figure 11 A-11B) from all elution holes is analyzed on plate gel.Running electrophoretic mobility is separated in HLS The evidence of compression is visible in fraction #2 (that is, segment 10-48.5kb is migrated altogether and found together in fraction #2, and in grade Divide in #1 without discovery DNA).Therefore, because compression phenomena will be seen that in fraction #2 greater than 10kb's under these conditions All DNA.Fraction #5 and #6 contain the segment that range is 1-2kb.

Bibliography

La Spada A.R. and Taylor, J.P., Repeat expansion disease:progress and puzzles in disease pathogenesis.Nature Reviews Genetics 11:247-258.

Nolin S.L. et al., Expansion of the Fragile X CGG Repeat in Females with Premutation or Intermediate Alleles.Am.J.Hum.Genet.72:454–464,2003.

6-50 unaffected 18-150bp

60-200 " pre- mutation " 180-600bp

Full mutation > 200 > 600bp

Suh, E.R. et al., Semi-automated quantification of C9orf72 expansion size reveals inverse correlation between hexanucleotide repeat number and disease duration in frontotemporal degeneration.Acta Neuropathol 130(3):363–372,2015.

The impacted 300-3800 (1800-22800bp) of unaffected 2-8 (12-48bp)

To presented in present patent application publication or other files (including but not limited to patent, patent application, paper, Webpage, books etc.) any and all references be all incorporated herein in its entirety by reference.

There have been described herein the exemplary embodiments of equipment, system and method.As other places indicate, these implementations Example being merely to illustrate property purpose description rather than it is restrictive.Other embodiments are possible and are covered by the disclosure that this will It is apparent from teaching contained herein.Therefore, the range of the disclosure and range should not be limited by any of above embodiment, And it should be limited according only to the claim and its equivalent that the disclosure is supported.In addition, the embodiment of present disclosure can be with It can also include from any and all of any other disclosed mthods, systems and devices including mthods, systems and devices Element, any and all elements including corresponding to molecule processing.In other words, from one or the other disclosed embodiment Element can be exchanged with the element from other open embodiments.Furthermore it is possible to remove one or more of the disclosed embodiments A feature/element, and still result in the theme that can be applied for a patent and (and therefore, lead to more implementations of this theme disclosure Example).Correspondingly, first by specifically lacking the one or more of system disclosed in such prior art, device and/or method Some embodiments of part/feature, present disclosure can be different in patent from one and/or another reference/prior art. In other words, negative limitation may include to the claim of some embodiments, specifically to exclude one or more element/spies Sign leads to the embodiment different in patent from including this category feature/element prior art.