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Trace and complex DNA amplification method

A complex and trace technology, used in complex DNA amplification and trace fields, can solve the problems of amplification failure, non-specific amplification, loss, etc., and achieve the effect of good specificity, good consistency, and reduced tripping.

Inactive Publication Date: 2013-06-05
SHANGHAI GREENGLOBE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]However, MDA / RCA technology also has some problems. Although it has high amplification efficiency and fidelity, it still has defects when used for single cell amplification, which affects the Its popularization and application in research and clinical practice mainly includes the following aspects: ① prone to amplification bias; ② prone to selective amplification or allele dropout, and allele selective amplification (Preferential Amplification, PA) and Allele Drop Out (ADO) are common problems in the process of single-cell PCR, and are also the key to misdiagnosis; ③ Abnormal amplification or loss of microsatellites often occurs; ④ Easy contamination or non-specific amplification, Due to the high sensitivity of MDA / RCA, a very small amount of contamination will cause amplification failure
Based on the above reasons, MDA / RCA technology is not widely used now

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1, Amplification of trace and complex DNA above 1ng

[0046] Step 1, whole genome DNA denaturation

[0047] Preparation buffer A: including 400 mmol / L KOH, 10 mmol / L EDTA

[0048] Preparation buffer N: including 200 mmol / L HCl, 300 mmol / L Tris-HCl (pH 7.5)

[0049] Add 1 μl of freshly prepared buffer A to a pre-cooled 200 μl thin-walled PCR tube, and then add 1 ul of whole genomic DNA (usually 1~5ng, as low as a single human cell or bacterial genomic DNA). After 3 minutes of denaturation (add 1 minute more for each additional 5ng of genomic DNA; if the amount of original DNA is small, reduce the denaturation time or dilute buffer A), add 1 μl buffer N and mix well.

[0050] Step 2, adding trehalose (TRE)

[0051] Quickly add 5 μl of 1.2M TRE, mix and place on ice.

[0052] The preparation method of 1.2M trehalose. Dilute with sterile DNA-free distilled water and then sterilize with a 0.22 micron filter (Millipore). Aliquots can be stored at -20°C for lon...

Embodiment 2

[0066] Example 2, High sensitivity, high fidelity, high uniformity, and high efficiency amplification of trace and complex DNA less than or equal to 0.5ng

[0067] Step 1, denaturation of trace and complex DNA

[0068] Referring to Example 1, for ≤ 0.5ng of pre-purified and extracted trace and complex DNA samples, dilute buffer A and buffer N described in Example 1 with 8 times of water, and refer to the denaturation method described in Example 1, DNA Denature in diluted buffer for 8 min.

[0069] When the sample is less than or equal to 0.1ng, the reduction in denaturation time is about one minute when the added DNA is reduced by one level.

[0070] Step 2, adding trehalose (TRE)

[0071] Quickly add 5 μl of 1.2M TRE, mix and place on ice.

[0072] Step 3, perform amplification

[0073] Then add the WPA reaction mixture described in Example 1 to the reaction system in step 2, turn it upside down several times to mix, add 1 μl of the modified phi29 DNA polymerase (concen...

Embodiment 3

[0076] Example 3, Amplification of Whole Cell DNA from Less Than 10 Cells

[0077] Step 1, Genomic DNA Denaturation

[0078] Preparation of denatured cell lysate: including 400mmol / L KOH, 10mmol / L EDTA, 100mmol / L DTT

[0079] Adjust the volume of cells (including corresponding solutions) to 1 μl, add 1 μl of cell lysate, place on ice, mix, and keep for 5 minutes. Then 1 μl of buffer N was added.

[0080] Step 2, adding trehalose (TRE)

[0081] Quickly add 5 μl of 1.2M TRE, mix and place on ice.

[0082] Step 3, perform amplification

[0083] Then add the WPA reaction mixture described in Example 1 to the reaction system in step 2, turn it upside down several times to mix, add 1 μl of the modified phi29 DNA polymerase (concentration 1 μg / μl), mix and turn to the constant temperature platform, in React at 30°C for 16 hours.

[0084] Step 4, terminate the reaction

[0085] After the amplification is completed, place the PCR reaction tube in a 70°C water bath for 20 minut...

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Abstract

The present invention relates to a high sensitivity, high fidelity, high uniformity and high efficiency amplification technology of trace and complex DNA. The technology system is constant temperature DNA amplification optimized by high concentration trehalose and guided by DNA polymerase, wherein balanced and efficient amplification of DNA in different regions of the whole genome or a DNA library sequence can be maintained in the context of effective inhibition on non-specific amplification by the amplification system. The method has the following characteristics that: sensitivity is high; balanced amplification of different region sequences can be maintained, and the amplification product has high fidelity (minimum deviation) and high uniformity; and the total reaction volume has high flexibility, and can be conveniently adjusted according to requirements. In addition, the technology provides broad application prospects in single cell level and trace genome DNA amplification and gene detection, pretreated function genome associated DNA library (spectrum) analysis, and other fields.

Description

Technical field [0001] The present invention involves an amplification method of a trace and complex DNA, especially the high -sensitivity, high -efficiency, high -efficiency amplification technology of a trace DNA, or single -cell or trace cell whole genome DNA, and seaweed sugarApplications in the allure DNA amplification. Background technique [0002] The detection of trace template DNA is a problem that is urgently needed to solve in many fields.Because the amount of DNA is insufficient, it is often unsuccessful with existing technical means.The development and application of PCR technology make the DNA analysis of trace and even a single cells possible, which has greatly promoted the development of forensic medicine, paleontology, molecular diagnosis and molecular pathology.But even for very sensitive PCR technology, the amount of DNA provided by many materials can only be used for one or several PCR reactions.On the other hand, PCR is specially focused on specific short (ge...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 潘星华蔡勇平
Owner SHANGHAI GREENGLOBE BIOTECH
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