CN1257179C - Method for achieving human DNA using adhesive tape - Google Patents
Method for achieving human DNA using adhesive tape Download PDFInfo
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- CN1257179C CN1257179C CN 01134233 CN01134233A CN1257179C CN 1257179 C CN1257179 C CN 1257179C CN 01134233 CN01134233 CN 01134233 CN 01134233 A CN01134233 A CN 01134233A CN 1257179 C CN1257179 C CN 1257179C
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- adhesive tape
- dna
- epithelial cell
- chelex
- suspension
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Abstract
The present invention discloses a method for extracting high-quality DNA by utilizing an adhesive tape to directly or indirectly obtain epithelial cells of human bodies, which belongs to the field of molecular biology. The method does not need to use proteinase K and has the advantage of rapid, simple and reliable operation on a method for extracting fingerprints on the surface of human bodies and a DNA extraction method. In addition, the method is suitable for sieving, identifying and determining the individual identification of medical jurisprudence, genetics, the identification in disputed paternity, etc. and the hereditary characteristics comprising hereditary diseases.
Description
Technical field
The present invention relates to biology field, specifically, the present invention relates to the preparation method and its usage of human DNA.
Background of invention
The hereditary material DNA that everyone carries varies, and dna polymorphism is enough to differentiate in a large amount of crowds individual.The dna typing technology is that the individuality that the inheritance from the people carries out is differentiated, and its tolerance range is far beyond the additive method height.
Modern paternity test and medical jurisprudence identify many with STR (STR) as the genetics mark.STR (STR) is distributed widely on 23 pairs of karyomit(e)s of people, and 100 dna fragmentations to hundreds of base pairs length by the base of 2-7 tumor-necrosis factor glycoproteins is formed have the polymorphism of height in the crowd; Utilize polymerase chain reaction (PCR) the technology target dna that can increase in a large number, rapidly and specifically.The sample of denier also can be detected behind pcr amplification, and the result can meet medicolegal requirement fully.
In recent years, can on DNA or gene level, diagnose increasing inherited disease.With Duchenne type muscular dystrophy is that example describes.Duchenne type muscular dystrophy is one group of heredodegeneration's disease that is primary in muscle, and sickness rate is 1/3500 life birth boy baby, and main clinical characteristics is the unable and atrophy of carrying out property of skeletal muscle of getting involved.Modern molecule genetics research proves that Duchenne type muscular dystrophy is by Xp on the X chromosome galianconism
21-Xp
223Due to the genetic flaw of sequence, this fragment gene is being controlled skeleton composition dystrophin (dystrophine) synthetic of muscle cell membrane, this gene is very huge, about 2.4Mb, contain 79 exons, 78 introns are in case so huge deletion mutantion of gene producer or point mutation all can have a strong impact on the synthetic of proteins encoded.The Duchenne type muscular dystrophy that studies show that the overwhelming majority is that another is at 3 of gene ' end 45-55 exon region because due to the deletion mutantion, these deletion mutantions have two districts occurred frequently, and one at 5 of gene ' end 1-7 exon region.Utilize round pcr can directly detect these deletion mutantions, thereby to the absence type patient or have genetic flaw person and diagnose.
For everyone, DNA is constant substantially throughout one's life, and the DNA that human body is organized everywhere except that sperm, ovum is mostly identical, so the DNA sample can be taken from: blood, seminal fluid, saliva, urine, hair, tooth, nail, bone, mouth epithelial cells, amniotic fluid even scurf.But the several method of gathering human DNA at present all has weak point.
For example, blood sampling is the sampling method of medical jurisprudence evaluation and medical diagnosis on disease routine, but the blood collection procedure complexity limits various.For example, the blood sample collection person must be professional medical matters personage, as doctor or nurse; The person of being sampled has the sensation even the feared state of mind of uncomfortable or pain; The person of being sampled is if any disease, as hepatitis, acquired immune deficiency syndrome (AIDS) or other transmissible diseases, just the practitioner has anxiety infected in the blood sample treating processes so.In addition, based on age, health, religion, law or other reasons, blood sample may be difficult to obtain.
Summary of the invention
The invention reside in provides a kind of simple and effective method of obtaining human DNA, and the examination of carrying out individual evaluation or hereditary feature analysis or genetic diseases further.The present invention also comprises the purposes that adhesive tape is used for directly or indirectly obtaining epithelial cell and/or its fragment.
The inventor has a spot of desquamation when finding at people's abutment solid surface or by fingerprint, can become the source of DNA.Fingerprint is the material evidence of normal collection during medical jurisprudence is identified, with fingerprint as the source of the DNA simple effective method of can yet be regarded as.Therefore use adhesive tape directly or indirectly to obtain the escutcheon at fingerprint or other position of health, take this to obtain epithelial cell, therefrom extract human DNA then and have very much practical significance and a using value.Adopt DNA can be used for individual differentiate and hereditary feature comprises screening, the evaluation and definite of inherited disease.
Method of the present invention comprises the steps:
(a) with the sticking directly or indirectly epithelial cell of getting human body of adhesive tape;
(b) in Chelex-100 suspension in 55 ℃-65 ℃, preferred 56 ℃-60 ℃ the insulation 2 hours to spending the night, for example be incubated 4 hours, 6 hours or 12 hours;
(c) boiling water bath 6-10 minute, preferred 7-8 minute;
(d) change with per minute 3000-13000, preferably the centrifugal 3-5 of speed minute of the 6000-13000 commentaries on classics, it was standby to get supernatant liquor.
The present invention uses adhesive tape to obtain the human body epithelial cell directly or indirectly, and extracting human DNA and carry out dna typing successfully therefrom is used for that individualities such as genetics, medical jurisprudence, paternity test are identified and hereditary feature comprises screening, the evaluation of inherited disease and determines.
Brief description of drawings:
Fig. 1 is the STR collection of illustrative plates of embodiment 1.Wherein left side figure is certain volunteer's fingerprint sample result, and right figure is this volunteer's blood sample result;
Fig. 2 is the STR collection of illustrative plates of embodiment 2.Wherein left side figure is certain volunteer's fingerprint sample result, and right figure is this volunteer's blood sample result;
Fig. 3 is the electrophoretogram of embodiment 3.Fig. 3 A is depicted as the electrophoresis result of certain volunteer's saliva sample behind exon 3, exon 43, exon 48 and exon 51 these four pairs of exon primer amplifications; Fig. 3 B is this volunteer's fingerprint sample electrophoresis result, swimming lane M is the molecular weight contrast, swimming lane N is the PCR negative control, swimming lane 1 is the exon 3 amplified production, swimming lane 2 is exon 43 amplified productions, swimming lane 3 is exon 48 amplified productions, and swimming lane 4 is exon 51 amplified productions, and the PCR the primer as shown in Table 1.
Embodiment
Method of the present invention comprises the steps:
(a) with the sticking directly or indirectly epithelial cell of getting human body of adhesive tape;
(b) in Chelex-100 suspension in 55 ℃-65 ℃, preferred 56 ℃-60 ℃ the insulation 2 hours to spending the night, for example be incubated 4 hours, 6 hours or 12 hours;
(c) boiling water bath 6-10 minute, preferred 7-8 minute;
(d) change with per minute 3000-13000, preferably the centrifugal 3-5 of speed minute of the 6000-13000 commentaries on classics, it was standby to get supernatant liquor.
Method of the present invention also preferably includes:
To be stained with epithelial adhesive tape with prior cleaning and disinfectant shear tool such as scissors shreds.
Method of the present invention also preferably includes:
After step (b) adds Chelex-100 suspension, before temperature is bathed with per minute 100-3000, preferred 2000 times hunting of frequency 10-20 second.
Method of the present invention also preferably includes:
Step (b) temperature bathe finish after with per minute 100-3000, preferred 2000 times hunting of frequency 10-20 second.
Method of the present invention also preferably includes:
After step (c) boiling water bath is finished with per minute 100-3000, preferred 2000 times hunting of frequency 10-20 second.
Above-mentioned Chelex-100 suspension is preferably freshly prepared 2-10%, preferred 4-8%, and more preferably 5% Chelex-100 suspension, its consumption is the 50-500 microlitre, preferred 100-300 microlitre, more preferably 200 microlitres.
In the method for the present invention, described epithelial cell can be from other position of finger, palm, toe, sole or health.
In the method for the present invention, described epithelial cell preferably tester's finger tripe part directly is pressed in obtain on the adhesive faces of adhesive tape or utilize that adhesive tape is sticking to be got the fingerprint that is retained on the body surface and obtain.
In the method for the present invention to the matrix of used adhesive tape without any restriction, it can be plastics or papery, can be transparent or nontransparent.
The described epithelial cell of context of the present invention comprises epithelial fragment.
Directly obtain the method for fingerprint: the finger tripe part that will point or other position of health such as palm, toe, sole etc. stay the escutcheon at fingerprint or other position directly firmly by on the mucilage glue surface that is pressed in adhesive tape.
Obtain the method for fingerprint indirectly: utilize the adhesive-layer of adhesive tape will be retained in object, take off as the fingerprint of slick wood, glass and stainless steel surface or the escutcheon of other body part.
Below be that example is described the process that the present invention preferably extracts DNA with the adhesive tape that is stained with fingerprint, and utilize the DNA that is obtained to diagnose the Duchenne muscular dystrophy by pcr amplification.
The adhesive tape that is stained with fingerprint part with cleaning in advance and shredding through the medical scissors of autoclaving and disinfection by ultraviolet light, is moved into centrifuge tube; Add 100-300 microlitre 4-8% Chelex-100 (Bio-Rad, UAS) suspension is with the hunting of frequency 10-20 second of per minute 100-3000 time; 56-60 ℃ is incubated 2 hours to spending the night; Again with hunting of frequency 10-20 second of per minute 100-3000 time; Boiling water bath 8-10 minute then, after finishing with hunting of frequency 10-20 second of per minute 100-3000 time; Change with per minute 6000-13000 at last the centrifugal 3-5 of speed minute, supernatant liquor moved in the new centrifuge tube to be stored in-20 ℃ of refrigerators standby.
The foundation of dna fingerprinting
Obtain after the DNA by method of the present invention, increase with the AmpF1 STR test kit of PE company, amplified production uses ABI PRISM310 Genetic Analyzer to analyze the back and produces dna fingerprinting.
In addition, for carrying out the screening purpose of genetic analysis or genetic diseases, after as above obtaining DNA, also can followingly carry out pcr amplification by the inventive method.For example, at Duchenne muscular dystrophy, can followingly increase:
Select four exon (Beggs of four pairs of primer amplification disappearance focuses in Duchenne type muscular dystrophy sudden change district occurred frequently, A.H.et al (1990) Detection of 98% ofDMD/BMD gene deletions by polymerase chain reaction.Hum Genet.86:45-48), this pcr amplification the primer is as shown in the table.Wherein a pair of 5 of the gene ' end that is positioned at, three pairs are positioned at 3 of gene ' end in addition, are respectively exon 3, exon 43, exon 48 and exon 51, and amplified production length is respectively 410bp, 357bp, 506bp and 388bp.Gel electrophoresis analysis is carried out to amplified production in the amplification back, with examination and the diagnosis of carrying out disease.
| Exon | Primer | Product length | |
| 3 | TCATCCATCATCTTCGGCAGATTAA | 410 | |
| CAGGCGGTAGAGTATGCCAAATGAAAATCA | |||
| 43 | GAACATGTCAAAGTCACTGGACTTCATGG | 357 | |
| ATATATGTGTTACCTACCCTTGTCGGTCC | |||
| 48 | TTGAATACATTGGTTAAATCCCAACATG | 506 | |
| CCTGAATAAAGTCTTCCTTACCACAC | |||
| 51 | GAAATTGGCTCTTTAGCTTGTGTTTC | 388 | |
| GGAGAGTAAAGTGATTGGTGGAAAATC |
The present invention has overcome the deficiency of existing method, still all easier on the DNA method for extracting on the fingerprint extraction method.Extract body surface gained escutcheon and needn't use special special adhesive tape.With the fingerprint is example, only need refer to that the fingerprint of tripe part is enough, does not need to extract whole palm line.DNA extraction is also simpler simultaneously, does not need to use Proteinase K, and whole process is faster simple and reliable.
The DNA that the inventive method obtained is applicable to that individualities such as genetic analysis, medical jurisprudence evaluation, paternity test are identified and hereditary feature comprises screening, the evaluation of inherited disease and determines.
Below with concrete embodiment the present invention is described.
Embodiment 1: utilize adhesive tape directly to obtain fingerprint
One volunteer firmly by on the mucilage glue surface that is pressed in scotch tape, with cleaning in advance and shredding through the medical scissors of autoclaving and disinfection by ultraviolet light, moves into 1.5 milliliters centrifuge tube with the adhesive tape that is stained with the fingerprint part with the finger tripe of its forefinger; (Bio-Rad, USA) suspension vibrated at a high speed 15 seconds with the frequency of per minute 2000 times to add the Chelex-100 of 200 microlitres freshly prepared 5%; 56 ℃ of incubated overnight; Vibrated at a high speed 15 seconds with the frequency of per minute 2000 times, boiling water bath is 10 minutes then again, vibrates at a high speed 15 seconds with the frequency of per minute 2000 times again; At last the speed of changeing with per minute 13000 is centrifugal 3 minutes, supernatant liquor is moved into to be stored in refrigerator in the new centrifuge tube standby.After extracting DNA from this scotch tape as stated above, get 3 microlitres and use the AmpF1 STR test kit of PE company to carry out pcr amplification according to the explanation of manufacturers, amplified production obtains dna fingerprinting after analyzing with ABI PRISM 310 Genetic Analyzer, this volunteer's STR collection of illustrative plates is, the male sex, THO1:7/9, TPOX:8/11, CSF1PO:10/10, D3S1358:17/17, vWA:17/18, FGA:24/24, D5S818:9/13, D13S317:8/8; D7S820:10/11; The result (referring to Fig. 1) in full accord that this result measures with getting this volunteer's blood.
Embodiment 2: utilize adhesive tape to obtain fingerprint indirectly
Utilize adhesive tape to obtain a volunteer and stay three pieces of fingerprints on the wooden desktop, the adhesive tape that is stained with the fingerprint part with cleaning in advance and shredding through the medical scissors of autoclaving and disinfection by ultraviolet light, is moved into the centrifuge tube of 1.5 microlitres; (Bio-Rad, USA) suspension vibrated at a high speed 15 seconds with the frequency of per minute 2000 times to add the Chelex-100 of 200 microlitres freshly prepared 5%; 56 ℃ of incubated overnight; Vibrated at a high speed 15 seconds with the frequency of per minute 2000 times, boiling water bath is 10 minutes then again, vibrates at a high speed 15 seconds with the frequency of per minute 2000 times again; The speed of changeing with per minute 13,000 is centrifugal 3 minutes at last, supernatant liquor is moved into to be stored in refrigerator in the new centrifuge tube standby.After extracting DNA from this scotch tape as stated above, get 3 microlitres and use the AmpF1 STR test kit of PE company to carry out pcr amplification according to the explanation of manufacturers.Amplified production obtains dna fingerprinting after using ABI PRISM 310 Genetic Analyzer to analyze, this volunteer's STR collection of illustrative plates is, the male sex, THO1:8/9, TPOX:8/11, CSF1PO:10/12, D3S1358:14/16, vWA:16/18, FGA:18/21, D5S818:11/12, D13S317:10/10; D7S820:8/11; The result (referring to Fig. 2) in full accord that this result measures with getting this volunteer's blood.
Embodiment 3: the detection of disease gene
One volunteer firmly by on the mucilage glue surface that is pressed in scotch tape, with cleaning in advance and shredding through the medical scissors of autoclaving and disinfection by ultraviolet light, moves into 1.5 milliliters centrifuge tube with the adhesive tape that is stained with the fingerprint part with the finger tripe of its forefinger; (Bio-Rad, USA) suspension vibrated at a high speed 15 seconds with the frequency of per minute 2000 times to add the Chelex-100 of 200 microlitres freshly prepared 5%; 56 ℃ of incubated overnight; Vibrated at a high speed 15 seconds with the frequency of per minute 2000 times, boiling water bath is 10 minutes then again, vibrates at a high speed 15 seconds with the frequency of per minute 2000 times again; The speed of changeing with per minute 13,000 is centrifugal 3 minutes at last, supernatant liquor is moved into to be stored in refrigerator in the new centrifuge tube standby.After extracting DNA from this scotch tape as stated above, get 5 microlitre DNA and add PCR damping fluid (100mM Tris HCl pH8.3,500mM KCl) 3.75 microlitres, 25mM MgCl respectively
22 microlitres, 10mM dNTPs 2 microlitres, DMSO 1.25 microlitres, water 8.75 microlitres, PCR primer 2 microlitre and Taq polysaccharase 0.25 microlitre (5U/ microlitre), the PCR reaction volume is 25 microlitres, reaction conditions be 95 ℃ 3 minutes, then 30 circulations be 94 ℃ 1 minute, 60 ℃ of 1 minute and 72 ℃ 2-4 minute (the extension time, per 10 circulations increased by 1 minute), last 72 ℃ of extensions finished in 5 minutes.Amplified production the results are shown in Figure 3 through 2% gel electrophoresis and ethidium bromide staining.Swimming lane M is the molecular weight contrast among Fig. 3 B, swimming lane N is the PCR negative control, swimming lane 1 is the exon 3 amplified production, swimming lane 2 is exon 43 amplified productions, swimming lane 3 is exon 48 amplified productions, swimming lane 4 is exon 51 amplified productions, and the result (Fig. 3 A) that this result measures with getting this volunteer's saliva is in full accord.
Though being example with the fingerprint, embodiments of the invention are described, those skilled in the art is after reading present disclosure, can be from extracting DNA with sticking the getting other position of testee's health such as other surperficial escutcheon of palm, toe, sole or health of adhesive tape, be used for that individualities such as genetic analysis, medical jurisprudence evaluation, paternity test are identified and hereditary feature comprises screening, the evaluation of inherited disease and determine that this point is conspicuous.
The present invention is not subjected to the restriction of above-mentioned concrete text description, the present invention can claims make various distortion and change in the scope of generalized spirit and essence.These distortion and change are all within the scope of the present invention.
＜110〉Hong Kong Chinese University
＜120〉a kind of method of utilizing adhesive tape to obtain human DNA
<130>SPI010824-09
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Claims (13)
1. method of extracting DNA, this method comprises the following steps:
(a) utilize the sticking human body epithelial cell of getting of adhesive tape;
(b) adhesive tape is incubated 2 hours to spending the night in 55 ℃-65 ℃ in 2-10% Chelex-100 suspension;
(c) boiling water bath 6-10 minute;
(d) with the centrifugal 3-5 of speed minute of per minute 3000-13000 commentaries on classics, it was standby to get supernatant liquor.
2. method according to claim 1 also comprises: sheared the adhesive tape fragmentation that the instrument that cuts will be stained with the fingerprint part with prior cleaning and disinfectant before adding Chelex-100 suspension.
3. method according to claim 1 also comprises: before step (c) boiling water bath with hunting of frequency 10-20 second of per minute 100-3000 time.
4. method according to claim 1 also comprises: after step (d) boiling water bath with frequency oscillation 10-20 second of per minute 100-3000 time.
5. according to the described method of claim 1-4, wherein used Chelex-100 suspension is fresh preparation, and consumption is the 50-500 microlitre.
6. according to the described method of claim 1-4, wherein step (b) is for being incubated 2 hour to spend the night in 55 ℃-60 ℃ with adhesive tape in the Chelex-100 of 4-8% suspension.
7. method according to claim 5, wherein step (b) is for being incubated 2 hour to spend the night in 55 ℃-60 ℃ with adhesive tape in 5% Chelex-100 suspension.
8. method according to claim 1, wherein said epithelial cell is from other positions of finger, palm, toe, sole or body surface.
9. method according to claim 1, wherein said epithelial cell by other positions of tester's finger, palm, toe, sole or body surface directly by being pressed on the adhesive tape and obtain.
10. method according to claim 9, wherein said epithelial cell is directly obtained by being pressed on the adhesive tape by tester's finger tripe part.
11. method according to claim 1, wherein said epithelial cell are to use, and adhesive tape is sticking to be got the escutcheon at the finger, palm, toe, sole or other positions of body surface that are retained on the body surface and obtains.
12. method according to claim 11, wherein said epithelial cell are to utilize that adhesive tape is sticking to be got the fingerprint that is retained in body surface and obtain.
13. method according to claim 1, wherein the matrix of adhesive tape be plastics or papery, transparent or nontransparent.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01134233 CN1257179C (en) | 2001-10-26 | 2001-10-26 | Method for achieving human DNA using adhesive tape |
| HK03107311A HK1054946A1 (en) | 2001-10-26 | 2003-10-13 | A non-invasive method of isolation of human dna |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01134233 CN1257179C (en) | 2001-10-26 | 2001-10-26 | Method for achieving human DNA using adhesive tape |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1414007A CN1414007A (en) | 2003-04-30 |
| CN1257179C true CN1257179C (en) | 2006-05-24 |
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ID=4672348
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01134233 Expired - Fee Related CN1257179C (en) | 2001-10-26 | 2001-10-26 | Method for achieving human DNA using adhesive tape |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1257179C (en) |
| HK (1) | HK1054946A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8898036B2 (en) | 2007-08-06 | 2014-11-25 | Rosemount Inc. | Process variable transmitter with acceleration sensor |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100365013C (en) * | 2006-03-21 | 2008-01-30 | 西安交通大学 | Method for extracting ancient human remains matter DNA |
| CN108498103A (en) * | 2018-03-30 | 2018-09-07 | 李刚 | Fingerprint on site and its pollution-free dual extracting methods of DNA |
-
2001
- 2001-10-26 CN CN 01134233 patent/CN1257179C/en not_active Expired - Fee Related
-
2003
- 2003-10-13 HK HK03107311A patent/HK1054946A1/en not_active IP Right Cessation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8898036B2 (en) | 2007-08-06 | 2014-11-25 | Rosemount Inc. | Process variable transmitter with acceleration sensor |
Also Published As
| Publication number | Publication date |
|---|---|
| HK1054946A1 (en) | 2003-12-19 |
| CN1414007A (en) | 2003-04-30 |
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