DNA SEQUENCING

Introduction

DNA sequencing is the terminology used for the arrangement of nucleotide bases like A (Adenine), G (Guanine), C (Cytosine) and T (Thymine) in DNA. Better understanding of DNA sequence has its own application in each and every field of biology and medicine like forensic medicine, biotechnology, diagnostic and therapeutic technology and biology systematic.  DNA sequencing has paved way for number of research and development. DNA sequencing found its scope during human genome mapping project. The extended research includes mapping of plant, animal, microorganisms and human. With the help of two dimensional chromatography technology first evident DNA sequence was obtained during 1970s by academic scientist. Invention of automated process helped DNA sequencing process in greater speed.

History of DNA sequencing

The oldest nucleotide sequencing technique is RNA sequencing. Walter Fiers of University of Ghent, Belgium and his coworkers sequenced complete gene and genome of Bacteriophage MS2 during the period of 1972 to 1976. This was first landmark an evidence of RNA sequencing. Before this evidence in 1970, Frederick Sanger of University of Cambridge, England, Walter Gilbert and Allan Maxam of Harvard University introduced rapid method of sequencing the DNA. The DNA sequencing done by Walter Gilbert and Allan Maxam is referred as wandering spot analysis with the help of 24 base pairs. The most reliable method of DNA sequencing is Sanger method called Chain termination method in the year 1975. It is easier and reliable method of sequencing the DNA as it incorporates use of dideoxynucleotide triphosphates or ddNTPs. This ddNTP is chain terminator of DNA.

Challenges of DNA sequencing

The quality of information of first 15 to 40 bases is not clear. Similarly 700 to 900 bases information retrieval is not possible. To improve this quality of common challenge Base calling software is employed. This helps in easy estimation of quality to be used for quality trimming. At times DNA is cloned just before sequencing due to the presence of vectors or cloning vehicles. For instance PCR based cloning on basis of pyrosequencing does not require any cloning vector. Ampliseq and SeqSharp, methods of Sanger DNA sequencing which combines sequencing and amplification in one step helps in easy target of genes just before amplification occurs. Recent method of DNA sequencing can be performed in a single step as short nucleotides. The shorter the size of DNA fragment the more information can be retrieved.

Questions:

• What are major landmarks in DNA sequencing?
• Write short note on DNA amplification and Clonal selection.
• Describe Maxam Gilbert and Chain termination DNA sequencing methods.
• What are high throughput sequencing?