Sanger Method: DNA Sequencing

Sanger Method: Sequence Analysis

Principle: The Sanger method, also known as dideoxy sequencing, is a laboratory technique used to determine the order of nucleotide bases in a DNA molecule. It is based on the principle of enzymatic synthesis of DNA strands, which are terminated by the incorporation of dideoxynucleotides.

Sequence Analysis:

Step 1: Template Preparation

• The target DNA is amplified and prepared for sequencing
• The DNA is denatured to create single-stranded templates

Step 2: Primer Design

• A primer is designed to bind to the target DNA sequence
• The primer is labeled with a radioactive or fluorescent marker

Step 3: Sequencing Reaction

• The template, primer, and dideoxynucleotides are mixed with DNA polymerase
• The reaction is carried out in four separate tubes, each containing a different dideoxynucleotide (ddATP, ddCTP, ddGTP, and ddTTP)
• The dideoxynucleotides are incorporated randomly into the growing DNA strand, terminating synthesis at specific points

Step 4: Gel Electrophoresis

• The reaction products are separated by size using gel electrophoresis
• The gel is run in a specific direction, allowing the shorter strands to migrate faster than the longer ones

Step 5: Autoradiography

• The gel is exposed to X-ray film or a phosphorimager to detect the radioactive or fluorescent labels
• The resulting autoradiogram shows a series of bands, each representing a different DNA sequence

Step 6: Sequence Determination

• The sequence of the DNA is determined by reading the autoradiogram from bottom to top
• The sequence is written in the 5' to 3' direction
• The dideoxynucleotides are used to determine the sequence of the DNA by identifying the termination points

Advantages:

• High accuracy and efficiency
• Can be used to sequence large DNA fragments
• Relatively fast and inexpensive compared to other sequencing methods

Limitations:

• Limited to sequencing DNA fragments up to 1 kb in length
• Requires a significant amount of DNA template
• Can be affected by the quality of the DNA template and primers

Sanger Method: Sequence Analysis

• The Sanger method, also known as dideoxy sequencing, is a laboratory technique used to determine the order of nucleotide bases in a DNA molecule
• The method is based on the principle of enzymatic synthesis of DNA strands, which are terminated by the incorporation of dideoxynucleotides

Template Preparation

• The target DNA is amplified and prepared for sequencing
• The DNA is denatured to create single-stranded templates

Primer Design

• A primer is designed to bind to the target DNA sequence
• The primer is labeled with a radioactive or fluorescent marker

Sequencing Reaction

• The template, primer, and dideoxynucleotides are mixed with DNA polymerase
• The reaction is carried out in four separate tubes, each containing a different dideoxynucleotide (ddATP, ddCTP, ddGTP, and ddTTP)
• The dideoxynucleotides are incorporated randomly into the growing DNA strand, terminating synthesis at specific points

Gel Electrophoresis

• The reaction products are separated by size using gel electrophoresis
• The gel is run in a specific direction, allowing the shorter strands to migrate faster than the longer ones

Autoradiography

• The gel is exposed to X-ray film or a phosphorimager to detect the radioactive or fluorescent labels
• The resulting autoradiogram shows a series of bands, each representing a different DNA sequence

Sequence Determination

• The sequence of the DNA is determined by reading the autoradiogram from bottom to top
• The sequence is written in the 5' to 3' direction
• The dideoxynucleotides are used to determine the sequence of the DNA by identifying the termination points

Advantages

• The Sanger method is highly accurate and efficient
• It can be used to sequence large DNA fragments
• The method is relatively fast and inexpensive compared to other sequencing methods

Limitations

• The Sanger method is limited to sequencing DNA fragments up to 1 kb in length
• It requires a significant amount of DNA template
• The method can be affected by the quality of the DNA template and primers