Sanger Sequencing and Its Applications (recording)

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Change the dates for the second exam to April 1-2. A preparation guide will be sent out to assist students in reviewing key concepts and materials covered in the course.

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Sanger Sequencing Overview

Developed by biochemist Frederick Sanger in the 1970s, Sanger sequencing revolutionized the field of genetics by providing a reliable method for sequencing DNA.

It’s a technique for sequencing DNA by determining the precise order of nucleotides in a DNA molecule. This method is essential not only for understanding genetic information but also for applications in cloning and genetic modification.

Outputs from this process include text files of sequences that can be used for further analysis, as well as histogram representations of data from sequencing runs, which help visualize the distribution of different nucleotide sequences generated during the experiment.

Connection to Cloning:

• Sanger sequencing is crucial in the cloning process as it confirms that the cloned gene is accurate and free of mutations after PCR amplification. This step is critical because PCR can introduce errors due to the inherent error-prone nature of the DNA polymerase used, as it lacks 3' exonuclease activity, leading to the potential for incorrect base incorporation.

Importance of Sequencing

All genes discussed in biotechnology code for proteins, making sequencing vital for understanding genetic function and expression.

Mutations can lead to significant issues such as:

• Changes in reading frames that affect downstream protein coding sequences.

• Creation of early stop codons that can result in truncated, dysfunctional proteins, which may lead to various genetic disorders or malfunctions in cellular processes.

Mechanisms of Sanger Sequencing

Also known as chain termination sequencing or dideoxy sequencing, this method relies on the selective incorporation of modified nucleotides that terminate DNA strand elongation.

Uses dideoxynucleoside triphosphates (ddNTPs) in conjunction with standard deoxynucleotides (dNTPs) to stop the elongation of DNA strands at specific points, creating fragments of varying lengths that can be analyzed.

Requires:

• A single-stranded DNA template where the sequence to be analyzed is present.

• DNA polymerase to extend the primers and synthesize new DNA strands.

• Primers that anneal to the template, providing a starting point for synthesis.

• DNTPs for regular DNA synthesis.

• Labeled ddNTPs which allow for visualization of the termination events.

High Resolution:

• Refers to the methodology's ability to distinguish small differences in DNA fragment length, which is critical for accurately determining sequences, especially in complex genomes.

DNA Extension and Termination

Incorporation of ddNTPs stops the reaction because these molecules lack a 3' hydroxyl group necessary for further DNA strand extension.

By maintaining a higher concentration of normal dNTPs compared to ddNTPs, it allows random incorporation of ddNTPs during DNA synthesis, leading to a tailored mixture of DNA fragments of different lengths that can be used to decode the original template sequence.

Separation of DNA Fragments

Gel Electrophoresis is the primary method used for separating DNA fragments, particularly using polyacrylamide gels for high-resolution separation, capable of resolving fragments that differ by a single base pair.

Early techniques employed radioactive isotopes (e.g., P32) for imaging the separation results on X-ray film (autoradiograms), although this method has largely been replaced by fluorescent labeling techniques in modern applications.

Automated DNA Sequencing

Modern sequencing techniques have combined Sanger sequencing principles with cycle sequencing, enabling multiple reactions to run simultaneously in a single tube.

Capillary electrophoresis improves the efficiency and resolution of the separation process, allowing for quicker results.

Advantages:

• Requires less DNA template, making it more accessible for samples of limited quantity.

• Enhanced sensitivity and resolution facilitate fine distinctions between closely related sequences.

• The capability to run multiple samples simultaneously increases throughput for sequencing projects.

Cycle Sequencing

Developed in the 1980s, this method involves cycling through stages that closely resemble PCR to amplify the sequences being analyzed, greatly improving efficiency.

The process allows for the use of double-stranded DNA templates, which broadens the applicability of sequencing techniques.

Labels are attached to ddNTPs during this process to enhance clarity and detection of the resultant reaction products.

Practical Applications

Primarily used for confirming gene sequences identified through high-throughput sequencing methods and serving as a benchmark standard for accuracy.

Sanger sequencing remains the gold standard for accuracy in genetic sequencing, particularly in clinical settings where precise mutation identification is crucial.

The sequence verification process involves conducting multiple small-scale reactions for larger genes and employs a primal walking technique to derive full sequences effectively.

Analysis of Sequencing Results

Each band in the resulting gel represents a specific sequence derived from the DNA template, allowing for correlation with known sequences or gene databases to ascertain their identity and function.

Determining sequences involves finding complementary matches to the sequencing products, an essential step in both research and diagnostic applications.

Conclusion

Despite being an older technology compared to newer sequencing methods, Sanger sequencing remains a vital method for ensuring accuracy in genetic analysis. Its reliability and precision continue to warrant its application in various fields of biology, often outsourced to specialized companies due to the cost and complexity associated with maintaining the necessary technology.