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When two or more colonies are double-picked or two or more inserts are present in your cloning vector, multiple peaks will appear within the insert site even though the peaks prior to the insert may look clear. (See Figure 1) |
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| Figure1. Mixed
template-plasmid Insert contamination |
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When two or more fragments of different lengths have similar electrophoretic mobility due to secondary structure, compressed multi-peaks can be detected after a specific point. Sometimes these compressed peaks may occur at high G/C or high A/T content regions. (See Figure 2) |
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| Figure2. Compression |
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Multiple binding occurs when two or more peaks with good signal strength appear at different positions. This occurs when two or more templates are mixed or multiple priming sites are present in your DNA template. (See Figure 3) |
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Figure3. Multiple Binding
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Slippage is present when mixed multi-peaks occur downstream along an extended homopolymer region. If the homopolymer region is not properly paired during polymerization, subsequent base slippage can be the result. (See Figure 4a-d) |
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Figure4a. Homopolymer region (Poly-T)
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Figure4b. Homopolymer region (Poly-A)
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Figure4c. Homopolymer region (Poly-C)
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Figure4d. Homopolymer region (Poly-G)
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When normal sequence data shows one base with partially overlapping peaks or double peaks, there could be a mixed base. A mixed base may be caused by the presence of an SNP or point mutation. (See Figure 5) |
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Figure5. Mixed Base
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In sequencing data, low peaks appear under major peaks. This may be caused by presence of byproducts from primer synthesis or degradation of your primer. We suggest you re-synthesize the primers to mediate this problem. (See Figure 6) |
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| Figure6. N-1 primer |
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In frame shift mutations, minor peaks appear under major peaks at a specific point within the sequence. Frame shift mutation is similar to the N-1 primer scenario, but affects PCR products. (See Figure 7) |
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Figure7. Frame Shift Mutation (PCR samples)
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Repeat Sequence (Microsatellite) is a direct result from a set of bases that repeat consecutively followed by the sequencing data becoming mixed and unreadable. This repeat can affect the polymerization process or cause a secondary structure to be formed, consequently revealing the mixed sequencing data. (See Figure 8a-b) |
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Figure8a. Repeat Sequence (GCA)
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Figure8b. Repeat Sequence (TGC)
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Abrupt signal loss can be attributed to a strong secondary structure or single stranded conformational structures (G/C-Rich regions) in the DNA template. In the case of an abrupt signal loss, there can be a sudden stop or a rapid decline in the sequence read. (See Figure 9a-b) |
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Figure9a. Abrupt Signal Loss - Sudden Stop
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Figure9b. Abrupt Signal Loss - Rapid Decline
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