The process of determining order of nucleotides in a specific DNA molecule is known as DNA sequencing. DNA sequencing is almost used in all branches of biology including molecular genetics, virology, metagenomics, Medicine, forensic investigations etc. Sanger sequencing is the gold method in DNA sequence analysis. Sanger sequencing is based on a principle called chain termination method named after the Nobel laureate Frederick sanger. Sanger sequencing procedure involves following steps
- Amplification of specific region of interest from the whole DNA macro molecule.
- Purification of amplified product to remove primer dimers, left over dNTP’s, enzymes etc.
- Chain termination process using the amplified product. The chain termination process is very much similar to normal PCR, but in this process along with dNTP’s, fluorescently labelled ddNTP’s are incorporated in the reaction mixture in appropriate quantity. The ddNTP’s lack the 3’OH group for the extension of DNA molecule. This process is also known as cycle sequencing
- Purification of cycle sequencing product.
- Separation of fragments using automated sequencing machine or by gel electrophoresis.
- Analyzing the data and interpretation of results.
DNA sequencing method depends on many factors like length of the target, complexity of the specimen, knowledge about the region of interest and throughput. DNA sequencing can be used for wide variety of applications.
Denovo sequencing: it is generally used to sequence the DNA molecule of unknown information. To sequence the unknown DNA molecule, first the target DNA is made into piece and cloned into a vector. This allows amplification of unknown DNA sequence using the known primer sequences in the vector. Smaller fragments of less than 50 kb are usually preferred for this technology for efficient results.
Resequencing: resequencing is used when the information about the target sequence is known. Target region of DNA is sequenced and compared with known or reference sequence. It is generally used to identify SNP’s(single nucleotide polymorphisms), mutations, small indels.
Epigenetics: one of the important step in gene expression regulation is DNA methylation. The DNA methylation status determines many phenotypic attributes. A CpG island associated with methylation specific cytosine can be identified using sanger sequencing. This methylated cytosine is protected from bisulfite conversion, whereas non methylated cytosine is converted to uracil.
Thermofisher scientific 3500 genetic analyzer is one of the automated sequencing technologies used to separate the cycle sequencing fragments. It has powerful data collection software which helps in real-time analysis of data quality. It has two major applications 1) sequencing 2) fragment analysis. Sequencing helps to identify variations in the genome and fragment analysis helps to identify copy number variations, aneuploids, large indels.