Compositions and methods for nucleic acid amplification
A technology for nucleic acid and target nucleic acid, applied in the field of compositions and methods for nucleic acid amplification, capable of solving problems such as reducing the efficiency of nucleic acid amplification reactions
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Embodiment 1
[0139] Example 1: Nucleic Acid Amplification via Primers with Molecular Parts
[0140] In six different amplification reaction mixtures (1-6) containing the complementary sequence of the human epidermal growth factor receptor (EGFR) gene (complementary sequence is shown in Figure 4 The nucleic acid of A) is amplified using a forward primer and a reverse primer. Each of the six reaction mixtures contained Figure 4 One of the four forward primers ("EG5", "EG5A", "EG5U" or "EG5T") and one of the two reverse primers ("EG3" or "EG3A") shown in B, where different reactions Mixtures contain different combinations of forward and reverse primers. The combination of forward and reverse primers in each reaction mixture is shown in Figure 4 C, where each reaction mixture is entered into the table at the intersection of its corresponding forward and reverse primers.
[0141] The various forward and reverse primers differ from each other by the presence or absence of molecular moieti...
Embodiment 2
[0144] Example 2: Nucleic Acid Amplification Via Primers Having Molecular Parts With Nucleotides With Unnatural Bases
[0145] Genomic nucleic acid comprising the sequence of the human epidermal growth factor receptor (EGFR) gene was amplified using forward and reverse primers in six different amplification reaction mixtures. Each of the ten reaction mixtures (00, 0C, 0G, 10, 1C, 1G, 20, 2C, 2G, 30) contained Figure 5 One of the four forward primers ("EG21e5", "EG21e5U1", "EG21e5U2" or "EG21e5U3") and one of the three reverse primers ("EG21e3", "EG21e3_iso-dC" or "EG21e3_iso- dG"), where different reaction mixtures contained different combinations of forward and reverse primers. The combination of forward and reverse primers in each reaction mixture is shown in Figure 5 In B, each of the reaction mixtures is entered into the table at the intersection of its corresponding forward and reverse primers.
[0146] The various forward and reverse primers differ from each other b...
Embodiment 3
[0149] Example 3: Nucleic Acid Amplification and Polymerase Types via Primers with Molecular Parts
[0150] Nucleic acid comprising the complementary sequence of the human epidermal growth factor receptor (EGFR) gene was amplified using forward and reverse primers in nine different amplification reaction mixtures. Each of the nine reaction mixtures (1-9) contained forward and reverse primers with molecular moieties at their 3' ends (e.g. Figure 6 As shown in A, the forward primer contains the molecular part of the sequence "U", and the reverse primer contains the molecular part of the sequence "iso-dG T") and one or two polymerases (Taq, cloned Pfu, Deepvent), Wherein different reaction mixtures comprise different polymerases (or combinations of polymerases) and / or different concentrations of polymerases. The polymerase and polymerase concentrations in each reaction mixture are shown in Figure 6 in B. The polymerases tested included a cloned Pfu polymerase control, Deep v...
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