Watson and Crick’s discovery of the DNA Double Helix structure in 1953 sprang the scientific community into life. With a few years attempts were being made to replicate the DNA sequence. By knowing the sequence of the nucleotides in the DNA, the location of the genes and mutations across populations in different species would be known. In 1970’s, two methods were independently developed to sequence DNA. The American team, lead by Maxam and Gilbert, used a “chemical cleavage protocol”, while the English team, lead by Sanger, developed the “chain termination method”. Even though both teams shared the 1980 Nobel Prize, Sanger’s method has survived the test of practicality and became the gold standard.

METHOD:

Sanger Method utilizes 2′, 3’-dideoxynucleotide triphospates (ddNTPs). ddNTPs are different from deoxynucleotides (dNTP) by the having a hydrogen atom at to the 3′ carbon instead of a hydroxyl group. ddNTPs terminate DNA chain elongation because they cannot form a phosphodiester bond with the next dNTP.

The double stranded DNA sequence is converted to a single stranded DNA sequence by denaturing the double stranded DNA with NaOH. Sanger method requires a single stranded DNA to be sequenced, DNA polymerase, DNA primers, a mixture of ddNTP with its normal dNTP and the other three dNTPs (dCTP, dGTP, and dTTP).  For example, “G” tube contains all four dNTP’s, ddGTP and DNA polymerase, “A” tube: all four dNTP’s, ddATP and DNA polymerase and so on. The concentration of ddNTP is 1% of the concentration of dNTP. When the DNA polymerase is added, the polymerization will take place and will terminate whenever a ddNTP is incorporated into the growing strand. Once the reactions in all the 4 tubes are complete, the DNA is denatured for electrophoresis. The contents of each of the four tubes are run on separate lanes on a polyacrylmide gel. Then the gel is then exposed to X-Ray for reading.  Smaller fragments travel faster and farther than the larger molecules due to their light molecular weight. When the reactions from the four tubes are combined on one gel plate and the bases are read from the bottom up, 5′ to 3′ sequence of the strand complementary to the sequenced strand can be read.

ADVANCES:

Automated Sequence: Sanger’s single radioactive dye where replaced with four fluorescent dyes for the 4 ddNTPs and the reactions are performed in a single tube containing all four ddNTPs, each labeled with a different color dye. Then the gel electrophoresis is done on a single lane instead of four and the DNA sequence is read using a chromatogram.

Microfluidic Sanger sequencing: Here the sequential steps from Sanger method are integrated on a wafer-scale chip using nanoliter-scale sample volumes. It generates long and accurate sequences, while overcoming many shortcomings of the conventional Sanger method.

Capillary Sanger sequence: It eliminated the use of gels. Instead, semi-liquid polymer was injected into the capillary before each run. Automated injection of samples allows hands-off operation through runs and amount of DNA sample required is reduced. Each run requires less than 3 hours.

The Human Genome Project: The 13 year long project of decoding the human genome was completed in 2003. Sanger sequencing technique was one of the methods used to sequence the DNA.

January 17th, 2014

Posted In: Uncategorized

Tags: , , ,

Leave a Comment