Primer Walking on Both DNA stands
DNA sequencing will start with two universal primers & continuously use primers derived from the end of the newly determined DNA sequences until the whole insert/fragment is fully sequenced on both ends. If there are any ambiguities in the sequence, we will design new primers to resolve them.
Description
- Primer walking double strand- Primer design, synthesis, and sequencing, the final alignment of data (Per Base)
- Primer walking double strand for AT or GC rich sequences-Primer design, synthesis, and sequencing, the final alignment of data (Per Base)
Some common applications of primer walking on both DNA strands include:
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Gene Sequencing: Primer walking allows for the step-by-step determination of the nucleotide sequence of a gene of interest. By extending primers on both the forward and reverse strands, researchers can obtain the complete coding sequence and regulatory regions of the gene.
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Genome Assembly: In large-scale sequencing projects, such as whole-genome sequencing, primer walking can be employed to validate and bridge gaps between contigs. By designing primers in regions flanking contigs, researchers can walk along the genome, filling in missing sequence information and ensuring accurate assembly.
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Characterization of Genetic Variations: Primer walking can be used to identify and validate genetic variations, such as single nucleotide polymorphisms (SNPs) or small insertions/deletions (indels), in a specific genomic region. By extending primers on both strands, researchers can compare the sequence information to reference sequences and identify variations associated with specific traits or diseases.
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Validation of Next-Generation Sequencing (NGS) Data: NGS technologies produce vast amounts of sequence data, but errors or biases can occur during the sequencing process. Primer walking can be used to validate and verify specific regions of interest identified through NGS. By obtaining the sequence through independent means, researchers can confirm the accuracy of NGS results.
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Investigating Structural Variations: Primer walking can aid in the characterization of structural variations, such as inversions, duplications, or translocations, by obtaining precise breakpoints and determining the organization of the affected genomic regions.
Overall, primer walking on both DNA strands is a versatile method used in DNA sequencing to obtain accurate and complete sequence information, validate sequencing results, and characterize genetic variations and structural variations in targeted genomic regions.
