Question

Briefly explain how Di-deoxy sequencing works.

What is another technique Di-deoxy sequencing is based on/similar to?

How do you determine the sequence if you run the reactions by conventional electrophoresis?

How does the automated version of Di-deoxy sequencing differ from using conventional gel electrophoresis?

What do you need to include in the reaction in order to determine where sequencing will begin?

Answer 1

-DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. The most popular method for doing this is called the dideoxy method or Sanger method.Sanger sequencing is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides.
In Sanger sequencing, the DNA to be sequenced serves as a template for DNA synthesis. A DNA primer is designed to be a starting point for DNA synthesis on the strand of DNA to be sequenced. Four individual DNA synthesis reactions are performed. The four reactions include normal A, G, C, and T deoxynucleotide triphosphates (dNTPs), and each contains a low level of one of four dideoxynucleotide triphosphates (ddNTPs): ddATP, ddGTP, ddCTP, or ddTTP. The four reactions can be named A, G, C and T, according to which of the four ddNTPs was included. When a ddNTP is incorporated into a chain of nucleotides, synthesis terminates. This is because the ddNTP molecule lacks a 3' hydroxyl group, which is required to form a link with the next nucleotide in the chain. Since the ddNTPs are randomly incorporated, synthesis terminates at many different positions for each reaction.

-The most recent set of DNA sequencing technologies are collectively referred to as next-generation sequencing.next-generation sequencing is kind of like running a very large number of tiny Sanger sequencing reactions in parallel.

-Following synthesis, the products of the A, G, C, and T reactions are individually loaded into four lanes of a single gel and separated using gel electrophoresis, a method that separates DNA fragments by their sizes. The bands of the gel are detected, and then the sequence is read from the bottom of the gel to the top, including bands in all four lanes.

For instance, if the lowest band across all four lanes appears in the A reaction lane, then the first nucleotide in the sequence is A. Then if the next band from bottom to top appears in the T lane, the second nucleotide in the sequence is T, and so on. Due to the use of dideoxynucleotides in the reactions, Sanger sequencing is also called "dideoxy" sequencing.

-The critical difference between Sanger sequencing and NGS is sequencing volume. While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run. This high-throughput feature makes it very cost-effective when sequencing a large amount of DNA.

-A primer, which is a short piece of single-stranded DNA that binds to the template DNA and acts as a "starter" for the polymerase.  The synthesis of a primer is necessary because the enzymes that synthesize DNA, which are called DNA polymerases, can only attach new DNA nucleotides to an existing strand of nucleotides.