Asymmetric PCR amplification method, special primer and application thereof
The application patent number is CN2004 10056866.0.
Patent application date: August 26th, 2004.
Asymmetric PCR amplification method, special primer and application thereof
Publication (announcement)No. CN 1629305
Publication date (announcement) June 22, 2005
Category chemistry; metallurgy
Certification date
right of priority
Application (patent right) for Beijing Boao Biochip Co., Ltd.; Tsinghua University
Address 102206 No.8, Life Science Park Road, Changping District, Beijing
Inventor (designer) Zhang Zhiwei; Wang Wei; Zhu Lingxiang; Zhang Qiong; EPICA
International application
International announcement
date of entry
Patent Agent Beijing Jikai Intellectual Property Agency Co., Ltd.
Agent Guan Chang
abstract
The invention discloses an asymmetric PCR amplification method, a special primer and an application thereof, and aims to provide a PCR amplification method capable of simply and efficiently preparing a single-stranded amplification product. The asymmetric PCR primer provided by the invention comprises a plurality of pairs of PCR primer pairs, and is characterized in that a segment of oligonucleotide tail irrelevant to the target sequence to be amplified is added to the 5' end of one primer in each pair of primers. The asymmetric PCR amplification method provided by the invention comprises the following steps: 1) pre-denaturation; The first part of PCR amplification includes several denaturation, primer annealing and primer extension cycles; The second part of PCR amplification includes several denaturation and primer extension cycles. An oligonucleotide tail unrelated to the target sequence to be amplified is added to the 5' end of one primer in a PCR primer pair for primer extension. The asymmetric PCR amplification method of the invention has high single-strand yield, can perform single or multiple PCR amplification, and can be widely used for nucleic acid detection.
Sovereignty terms
1. An asymmetric PCR primer includes several pairs of PCR primers, and is characterized in that a segment of oligonucleotide tail unrelated to the target sequence to be amplified is added to the 5' end of one primer in each pair.
Direct sequencing of PCR products
Gel purification of PCR amplification target sequence
If the optimal PCR reaction conditions can not produce the required specific products, we can re-amplify them with new oligonucleotides, or separate different PCR products by gel electrophoresis, and then re-amplify each PCR product for sequence analysis. Fragments with different lengths can be separated by agarose gel electrophoresis:
1. When the fragment size is 80- 100bp, 3%NuSieve 1% common agarose or polyacrylamide gel can be used for separation.
2. Slices containing PCR fragments from South Africa were cut from the gel.
3. Add 50∽ 100μlTE to soak the film, which can be repeatedly frozen or left for several hours to make DNA diffuse out of the gel.
4. Take a small amount (1-5%) for the second amplification. In order to obtain a pure single product, the amount of gel extract used for the second PCR amplification must be less than 65438±0 ng.
5. It has been found that agarose contains substances that inhibit the activity of Tab polymerase. Therefore, if it is necessary to re-amplify the fragment for Tab polymerase sequencing, it is best to separate it by acrylamide electrophoresis.
When PCR primers are used to amplify several related sequences with the same length, such as the same primer from several newly replicated genes, repetitive sequences or conservative signal sequences, electrophoretic separation can be improved by the following two different methods. 1). First, only one template was cut by endonuclease, and then the complete PCR product was purified by electrophoresis. 2) Electrophoresis system for separating amplification products with different nucleotide sequences. The next section will discuss the denatured formamide gradient gel system in this kind of system. At present, it is necessary to know the endonuclease sites in different PCR products in advance.
Direct sequencing of hybrids
When two alleles are different due to single site mutation, the heterozygous site can be found by direct sequencing with PCR primers. However, allele templates with several point mutations or short insertion/deletion fragments will be sequenced directly with a PC R primer, which will produce a composite sequencing ladder. There are four methods to determine several bulges and obtain the sequence of a single allele from heterozygotes: 1). Cloning and separating different templates; 2) Before sequencing, different templates were separated by electrophoresis using the difference of nucleotide sequences between templates; 3) Only one allele is started in the sequencing reaction; 4). Only one allele was amplified.
Preparation of sequencing template
Some problems related to the direct sequencing of PCR products are that after denaturation, the two strands of the amplified fragment can quickly combine, thus preventing the sequencing primer from annealing with its complementary sequence or preventing the extension of the primer-template complex. In the sequencing reaction, template chain recombination makes a small number of modules analyze and sequence, thus weakening the final sequencing band. In order to reduce this problem, single-stranded templates can be prepared by improved standard double-stranded DNA sequencing or PCR.
Double-stranded DNA template
There are two different methods to prepare templates for sequencing, and both methods have been used to determine the sequence of closed-loop double-stranded plasmid templates with valence of * * *. It is usually difficult to sequence PCR products by these methods, because short linear templates are easier to recombine than double-stranded plasmids. In both methods, PCR fragments can be purified by gel electrophoresis or rotary dialysis before electrophoresis.
1. At room temperature, the template was denatured in 0.2M NaOH for 5Min, frozen, and neutralized with 0.4 times of 5m ammonium acetate (pH7.5). Immediately precipitate DNA with four volumes of absolute ethanol. Adding sequencing buffer primers at suitable annealing temperature.
2. Keep the temperature at 95℃ for 5 minutes to denature the template, and quickly cool the centrifuge tube in an ice bath (or dry ice ethanol) to reduce chain recombination. Adding sequencing primers and allowing the reaction to reach an appropriate temperature. Sequencing primers are suitable to be added before or after denaturation.
Single strand DNA template
Using single-stranded template can avoid strand recombination in sequencing. Single-stranded templates can be prepared from double-stranded DNA templates by strand separation gel or PCR reaction. Single-stranded DNA fragments larger than 500bp can be separated from agarose gel, but it is not suitable for shorter single strands. Another different method for preparing single-stranded DNA is to use biotin-labeled primers in PCR reaction. Denatured PCR products were separated into two strands by avidin column, and only the biotin-labeled strand could be bound to the column. However, the simplest method is to use the improved PCR method to prepare the established single-stranded DNA. In this reaction (asymmetric PCR, asymmetric PCR), in the first 20-25 cycles, two amplification primers with asymmetric proportions produce double-stranded DNA, and when the restriction primers are used up, the following 5- 10 cycles produce ssDNA. Single-stranded DNA began to appear after about 25 cycles, and the limited number of primers was almost exhausted. After a short and rapid rise, ssDNA began to accumulate linearly as expected, when there was only one primer in the reaction. Different proportions of primers produce this form of ssDNA. Generally speaking, for the reaction system of 100μlPCR, the primer ratio is 50pmo:0.5pmol, and about 1-3pmol of ssDNA can be produced after 30 cycles. The yield of ssDNA can be estimated by the following methods: a). In the PCR reaction, 32P-dNTP was added in addition to the normal amount of dDNA. The reaction product of 10% was electrophoresed in a thin 3% NuSieve+ 1% ordinary agarose gel, dried and exposed together with the film. b)。 5% of the reactants were gelled, transferred to the membrane, and hybridized with oligonucleotides complementary to ssDNA as probes. SsDNA can not be quantified by EB staining, because ssDNA tends to form secondary structure, and the insertion of dyes in the template is random. The amplification efficiency of asymmetric primer ratio is lower than (70%)80-90% excess of two primers. In experiments, this can be compensated by increasing the number of PCR cycles. If the asymmetric PCR reaction can't produce enough ssDNA, we can try different primer ratios. B) add 5- 10 PCR cycles; C) Add more Tab polymerase (2U) in the last five cycles; d)。 Try the opposite asymmetric primer ratio. Sometimes, the opposite asymmetric primer ratio may produce different ssDNA yields. A limited number of PCR primers or internal primers were used to sequence the prepared single-stranded DNA, and incorporation sequencing or labeling primer sequencing were carried out by conventional methods. The prepared ssDNA chain may have discontinuous 5'- end, but it may be cut off at different sites near 3'- end, which is caused by premature termination during extension. However, for any primer used in the sequencing reaction, only intact ssDNA can be used as the sequencing template.
Recently, another method for preparing single-stranded nucleic acid templates for direct sequencing has been reported. The method includes adding phage promoter to PCR primer, transcribing RNA copy of PCR product, and then using reverse transcriptase without sequencing. However, the application of this method is greatly limited because of the addition of enzymatic reaction steps after amplification reaction and the limitation of using reverse transcriptase as sequencing enzyme.
Double-stranded DNA template
There are two different methods to prepare templates for sequencing, and both methods have been used to determine the sequence of closed-loop double-stranded plasmid templates with valence of * * *. It is usually difficult to sequence PCR products by these methods, because short linear templates are easier to recombine than double-stranded plasmids. In both methods, PCR fragments can be purified by gel electrophoresis or rotary dialysis before electrophoresis.
1. At room temperature, the template was denatured in 0.2M NaOH for 5Min, frozen, and neutralized with 0.4 times of 5m ammonium acetate (pH7.5). Immediately precipitate DNA with four volumes of absolute ethanol. Adding sequencing buffer primers at suitable annealing temperature.
2. Keep the temperature at 95℃ for 5 minutes to denature the template, and quickly cool the centrifuge tube in an ice bath (or dry ice ethanol) to reduce chain recombination. Adding sequencing primers and allowing the reaction to reach an appropriate temperature. Sequencing primers are suitable to be added before or after denaturation.
Single strand DNA template
Using single-stranded template can avoid strand recombination in sequencing. Single-stranded templates can be prepared from double-stranded DNA templates by strand separation gel or PCR reaction. Single-stranded DNA fragments larger than 500bp can be separated from agarose gel, but it is not suitable for shorter single strands. Another different method for preparing single-stranded DNA is to use biotin-labeled primers in PCR reaction. Denatured PCR products were separated into two strands by avidin column, and only the biotin-labeled strand could be bound to the column. However, the simplest method is to use the improved PCR method to prepare the established single-stranded DNA. In this reaction (asymmetric PCR, asymmetric PCR), in the first 20-25 cycles, two amplification primers with asymmetric proportions produce double-stranded DNA, and when the restriction primers are used up, the following 5- 10 cycles produce ssDNA. Single-stranded DNA began to appear after about 25 cycles, and the limited number of primers was almost exhausted. After a short and rapid rise, ssDNA began to accumulate linearly as expected, when there was only one primer in the reaction. Different proportions of primers produce this form of ssDNA. Generally speaking, for the reaction system of 100μlPCR, the primer ratio is 50pmo:0.5pmol, and about 1-3pmol of ssDNA can be produced after 30 cycles. The yield of ssDNA can be estimated by the following methods: a). In the PCR reaction, 32P-dNTP was added in addition to the normal amount of dDNA. The reaction product of 10% was electrophoresed in a thin 3% NuSieve+ 1% ordinary agarose gel, dried and exposed together with the film. b)。 5% of the reactants were gelled, transferred to the membrane, and hybridized with oligonucleotides complementary to ssDNA as probes. SsDNA can not be quantified by EB staining, because ssDNA tends to form secondary structure, and the insertion of dyes in the template is random. The amplification efficiency of asymmetric primer ratio is lower than (70%)80-90% excess of two primers. In experiments, this can be compensated by increasing the number of PCR cycles. If the asymmetric PCR reaction can't produce enough ssDNA, we can try different primer ratios. B) add 5- 10 PCR cycles; C) Add more Tab polymerase (2U) in the last five cycles; d)。 Try the opposite asymmetric primer ratio. Sometimes, the opposite asymmetric primer ratio may produce different ssDNA yields. A limited number of PCR primers or internal primers were used to sequence the prepared single-stranded DNA, and incorporation sequencing or labeling primer sequencing were carried out by conventional methods. The prepared ssDNA chain may have discontinuous 5'- end, but it may be cut off at different sites near 3'- end, which is caused by premature termination during extension. However, for any primer used in the sequencing reaction, only intact ssDNA can be used as the sequencing template.
Recently, another method for preparing single-stranded nucleic acid templates for direct sequencing has been reported. The method includes adding phage promoter to PCR primer, transcribing RNA copy of PCR product, and then using reverse transcriptase without sequencing. However, the application of this method is greatly limited because of the addition of enzymatic reaction steps after amplification reaction and the limitation of using reverse transcriptase as sequencing enzyme.