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US 2021/0079458 A1Published application
Amplification method of a single stranded dna
AssigneesNational Guard Health Affairs, King Saud bin Abdulaziz University for Health Sciences, King Abdullah International Medical Research Center
InventorsAtef Nehdi, Mohamed Boudjelal, Nosaibah Samman
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Abstract
A method for preparing a single stranded DNA by the polymerase chain reaction in high yield and purity. The method employ one regular primer and one modified primer at the 5′-end. The modified primer has a primer segment linked to an oligo- or polynucleotide segment through a 5′-5′-phosphodiester bond. Using the modified primer, the PCR product includes two complementary DNA strands having different lengths and separable from one another.
Claims20
1:
- A PCR method for the synthesis of a single stranded nucleic acid sequence, comprising:
- preparing a polymerase chain reaction comprising a template, a first modified primer, a second primer, a nucleotide triphosphate, and a DNA-polymerase,
- carrying out a plurality of amplification reactions by cycling the temperature of the reaction mixture between 20° C. and 100° C. to form product DNA strands, and
- isolating the product DNA strands;
- wherein the first modified primer comprises a first oligonucleotide segment of 10-50 nucleotides in length complementary to a target nucleic acid and having an extendable 3′-end and a 5′-end linked to the 5′-end of a second oligonucleotide segment of 10-100 nucleotides or a polynucleotide.
2 more elements
2:
- The method of claim 1, wherein the product DNA strands are isolated by denaturing electrophoresis.
3:
- The method of claim 1, wherein the template is a DNA strand.
4:
- The method of claim 3, wherein the DNA-polymerase is a thermophilic DNA-dependent DNA polymerase.
5:
- The method of claim 1, wherein the second oligonucleotide segment is an oligo-dA, or poly-dA.
6:
- The method of claim 5, further comprising:
- separating the product DNA strands from the reaction mixture by binding to oligo- or poly(dT) or (U) supported on a solid support.
7:
- The method of claim 6, further comprising:
- releasing the product DNA strands from the solid support by denaturation.
8:
- The method of claim 1, wherein the template is a RNA strand.
9:
- The method of claim 8, wherein the DNA polymerase is an RNA-dependent DNA-polymerase.
10:
- A PCR method for the synthesis of a single stranded nucleic acid sequence, comprising:
- preparing a polymerase chain reaction comprising a template, a first modified primer, a second primer, a nucleotide triphosphate, and a nucleic acid-polymerase,
- carrying out a plurality of amplification reactions by cycling the temperature of the polymerase chain reaction between 20° C. and 100° C. to form a reaction mixture comprising product DNA strands, and
- isolating the product DNA strands;
- wherein the modified primer comprises a first oligonucleotide segment of 10-50 nucleotides complementary to a target DNA template having an extendable 3′-end and a 5′-end linked to the 5′-end of a nucleotide or dinucleotide which is linked through its 3′-end to a 3′-end through 3′-3′ linkage to a second oligonucleotide segment of 10-100 nucleotides or a polynucleotide.
2 more elements
11:
- The method of claim 10, wherein the product DNA strands are isolated by denaturing electrophoresis.
12:
- The method of claim 10, wherein the template is a DNA strand.
13:
- The method of claim 10, wherein the DNA polymerase is a thermophilic DNA-dependent DNA polymerase.
14:
- The method of claim 10, wherein the second oligonucleotide segment is linked to oligo-dA, or poly-dA.
15:
- The method of claim 14, wherein the DNA strands are removed from the reaction mixture by a solid support modified by a fragment of oligo or poly(dT) or (U).
16:
- The method of claim 15, wherein the product DNA strands are isolated by denaturing electrophoresis.
17:
- The method of claim 10, wherein the template is a RNA strand.
18:
- The method of claim 8, wherein the DNA polymerase is RNA-dependent DNA polymerase.
19:
- A polynucleotide primer, comprising:
- a first oligonucleotide segment and a second oligonucleotide segment linked by 5′-5′-phosphodiester bond,
- wherein the first oligonucleotide segment comprises a 10-50 nucleotide sequence complementary to a target nucleic acid for amplification and the second oligonucleotide segment is an oligonucleotide of 10-99 residues or a polynucleotide.
20:
- The polynucleotide primer of claim 20, wherein the second oligonucleotide segment is oligo- or poly-dA, oligo- or poly-dT, oligo- or poly-dG, or oligo- or poly-dC.