Direct DNA Sequencing of Complementary DNA Amplified by the Polymerase Chain Reaction
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1. |
The use of 5′-end-labeled DNA-sequencing primers that are complementary to regions between the PCR primers (3 );
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2. |
Gel purification of amplified DNA to remove unwanted fragments and primer (4 );
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3. |
Spin columns for the separation of leftover primers from high mol wt material (5 , 6 );
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4. |
“Asymmetric” or “unbalanced” PCR priming to generate an excess of single strands during the initial amplification (7 );
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5. |
Addition of dimethylsulfoxide (DMSO) to sequencing reactions with short annealing times (8 ); and
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6. |
The use of several short, high-temperature, sequencing cycles (9 ).
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