The Sanger Method: Developed By Frederic Sanger
The Sanger Method was developed by Frederic Sanger. It was the first method developed to sequence the genome and the genetic code and is still the most commonly used method of DNA sequencing. In 1980 Sanger was awarded a nobel prize in chemistry for his work concerning DNA sequencing along with Paul Berg and Walter Gilbert who also contribuated in this major breakthrough. Since then the Dideoxy chain termination method has been highly developed and optimised.
To sequence DNA in term of the Sanger Method DNA first must be obtained in a single strand before it can be sequenced. Then a short oligonucleotide primer which is complementary to the 3' end of the template DNA must be annealed to the templete strand. The primer is necessary for the …show more content…
Both of these isotopes emit high energy beta particles which can blacken x-ray film. Sulfur-35 is most commonly used as it emits lower energy , less dangerous beta particles than phosphorus-3. Polyacrylamide gel electrophoresis is then used to seperate and analyse the polynucleotide chains in sizes ranging between 15 and 1000 nucleotides on the basis of size. The four mixtures of radiolabelled polynucleotides are transferred into separate wells according to the nucleotide type. The nucleotides are run through the gel at a voltage of 1500 volts. Due to the fact that DNA strands are negatively charged they are attracted to the positive anode and therefore move down the gel towards the anode. The DNA strands are separated on the basis their nucleotide type and on the basis of their size. Smaller oligonucleotides travel further down the gel and can be separated by as little as one nucleotide. Eventually they will indigilise on the gel with a radioactive band. The gel is dried and exposed to an x-ray film. The developed film is called a radiograph. Each of the radiolabelled oligonucleotide species is repesented by a dark black band on the radiograph. The DNA sequence can then be determined from this radiograph. The whole process separates these polynucleotides completmentary to the template strand. The 5'-3' DNA sequence can then be determined by reading from the bottom of the gel upwards one
To sequence DNA in term of the Sanger Method DNA first must be obtained in a single strand before it can be sequenced. Then a short oligonucleotide primer which is complementary to the 3' end of the template DNA must be annealed to the templete strand. The primer is necessary for the …show more content…
Both of these isotopes emit high energy beta particles which can blacken x-ray film. Sulfur-35 is most commonly used as it emits lower energy , less dangerous beta particles than phosphorus-3. Polyacrylamide gel electrophoresis is then used to seperate and analyse the polynucleotide chains in sizes ranging between 15 and 1000 nucleotides on the basis of size. The four mixtures of radiolabelled polynucleotides are transferred into separate wells according to the nucleotide type. The nucleotides are run through the gel at a voltage of 1500 volts. Due to the fact that DNA strands are negatively charged they are attracted to the positive anode and therefore move down the gel towards the anode. The DNA strands are separated on the basis their nucleotide type and on the basis of their size. Smaller oligonucleotides travel further down the gel and can be separated by as little as one nucleotide. Eventually they will indigilise on the gel with a radioactive band. The gel is dried and exposed to an x-ray film. The developed film is called a radiograph. Each of the radiolabelled oligonucleotide species is repesented by a dark black band on the radiograph. The DNA sequence can then be determined from this radiograph. The whole process separates these polynucleotides completmentary to the template strand. The 5'-3' DNA sequence can then be determined by reading from the bottom of the gel upwards one