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PCR Failures and Insights

• Initial reaction may fail, leading to unexpected results.

• A grad student observed a bigger band than expected in their gel for PCR result.

• The unexpected size was due to an incorrect assumption about primer length and product size.

Lesson from Mistakes

• Importance of analyzing failed experiments instead of discarding them.

• Many early PCR experiments led to failures, as researchers were still learning the technique.

• Distinction between getting no results versus unexpected but informative results.

Sanger’s Discoveries in Sequencing

• In a failed reaction, missing a crucial base provided information about the sequence.

• The absence of a 'C' indicated its position as the sixth base of the DNA sequence.

• Encouraged further experimentation by selectively omitting bases to uncover the sequence.

Method of Experimentation:

• New experiment set up by including 'C' and omitting 'T'.

• Successfully yielded a sequence of different bases, leading to DNA sequencing discovery.

• Result was akin to solving a puzzle by identifying bases one by one, resembling a game of hangman.

The Mechanics of DNA Sequencing

• Description of preparing reactions with normal bases and poisonous versions to halt the process.

• Utilizing both normal and toxic triphosphates to allow determination of the DNA fragment lengths.

• The connection between the length of fragments on a gel and the sequence of the DNA base.

• e.g., lack of a normal base leads to a shorter fragment.

Innovations in Sequencing Techniques

• Creating dideoxynucleotides (ddNTPs) to halt DNA synthesis by removing hydroxyl groups.

• Four dideoxynucleotides created for each base: A, T, C, G.

• Each reaction generates fragments that correspond to the length of the sequence where normal bases were incorporated versus poisonous.

Improvements in the Process:

• Initially, Sanger sequencing used gel electrophoresis for size differentiation of DNA fragments.

• Transition towards automation and more efficient detection methods, using colored dyes for better differentiation.

• Introduction of machines that can sequence a much larger number of bases rapidly and accurately.

Applications of DNA Sequencing

• Genome sequencing of whole organisms, using shotgun sequencing to assemble pieces of the genome from many fragments.

• Capability to explore phylogenetic relationships and genetic diseases through sequencing insights.

• Technologies like 23andMe utilizing sequencing to give personalized genetic information based on limited, valuable segments.

Implications in Research and Health

• Understanding of genetic diseases, tracking viral outbreaks, and studying evolutionary biology through sequencing.

• Practical applications in healthcare, such as identifying genetic predispositions or tracking mutations in viruses and bacteria.