Question

sanger sequencing, and how base identity (A,T,C,G) is detected using fluorescence. Can you explain what it means that the DNA is labeled with a fluorescent tag, and where that tag is located?

Answer 1

sanger sequencing:

It is a method of identifying the nucleotide bases and order of arrangement of these nucleotide bases on DNA.

It is called sanger sequencing as it was first development by Frederick Sanger and colleagues in 1977.

This is also called as chain termination or dideoxy method.

The principle behind this is utilization of specially designed nucleotides2', 3' ddNTP'S(Dideoxy nucleotide triphosphates) which acts as terminator molecules, stops chain elongation i.e. they do not form phosphodiester bond with next deoxynucleotide.

The ddNTP's designed have hydrogen atom bound to 3' carbon, rather than an hydrosyl(OH) group and have different fluorescence color

Overview of sanger sequencing:

1) using enzymatic synthesis of complementary polynucleotide chains, determine the sequence of single stranded DNA molecule

2) Terminate the above obtained chains at specific nucleotide positions

3) Separate by gel electrophoresis

4) Read DNA sequence

Steps in PCR:

• The DNA of interest or single strande DNA to be sequenced is subjected to PCR
• The above steps requires template(amplified sequence obtained from first PCR), Primer, polymerase and mixture of ddNTP's and dNTP's, double distilled water
• DNA pol used dNTP's to extend the DNA
• single DNA strand is elongated by random addition of dNTP's and ddNTP's
• ddNTP's terminates elongation, shorter or longer fragment depends on early or late addition of ddNTP's respectively.
• As the above steps is random, all possible lengths of DNA is obtained with terminal fluroscent labelled ddNTP
• Product or fragments obtained is denature and subjected to Gel electrophoresis
• Image the gel, the fragments are read from bottom to top, this represents the complementary sequence of DNA

Fluorescent labellin of DNA;

Fluorescent labelling is usually carried out by enzymatic reactions, that is they are enzymatically introduced in to DNA/cDNA by modified nucleotides

Organic flurophores are chemically introduced into nucleoside triphosphates or primers.

The flurophores selectively binds to the specific region or functional group on the target molecule.

Then it is incorporated using PCR amplification or using DNA polymerases or terminal nucleotide transferase

Fluroscent labelled DNA Tag:

This means the hybrid DNA with flurorescent molecule

The DNA is first constructed by gene and DNA of fluorescent protein

After transcritpion hybrid RNA and fluorescent is formed, the DNA of interest is attached to this hybrid DNA using an enzyme that can recognise the hybrid DNA.

Biotin or fluorescein are used as flurophores.