About Sequencing

[Sequencing]

 

Definition

Sequencing is an analysis of biopolymer such as DNA, RNA, and protein for finding out the exact structure (atomic-level). RNA sequencing is a process of ordering and lining the nucleotides in given structure. Although the RNA information can be acquired by observing DNA sequence, it has its own meaning. Protein sequencing can be conducted in various way, Edman degradation, Peptide mass fingerprinting, Protease digests, and Mass spectrometry. Through protein sequencing, it can be helpful to sequence target DNA by inferring the protein information.

 

DNA Sequencing

Similar with RNA sequencing, the DNA sequencing is an analysis of ordered nucleotide in DNA fragments.

First sequencing method which is used most broadly in the early stage is ‘Sanger sequencing’ that is designed by Frederick Sanger. The main feature of this method is the chain termination nucleotide, dideoxynucleotide triphosphates (ddNTPs). The extension of DNA strands is terminated by this nucleotide due to the lack of phosphatidiester bond in 3’ OH which links one nucleotide and next one. Thus the addition of this molecule generates many DNA fragments in various lengths. Then the fragments are separated by size using electrophoresis and each nucleotide in the end of each fragment can be detected by fluorescence.

As the methods are keep developed, in these days, the most genome data is analyzed more by pyrosequencing (or 454 sequencing) than Sanger sequencing. It enables faster analysis by using single-nucleotide addition (SNA) method. Due to the limitation in the numbers of nucleotides that can be paused during DNA elongation, the strands can be extended by adding more single nucleotides. And each nucleotide which is linked on the strand adds fluorescence gradually. The increment of its signal can be analyzed in the form of program, which shows the order of lettered nucleotides.

 

Human Genome Project

The goal of Human Genome Project is to sequence all existing human chromosome (about 3 billion nucleotides). If the whole genome is determined and analyzed, the every person could be diagnosed with personalized genome information and easy-detection of disease that can occur in future would be possible. And the extreme advance of medical science would be also possible with whole genome information.

Using those methods described above, many stages of Human Genome Project is completed successfully. With present information, much advanced biomedical research is possible. But still millions of repeat-rich heterochromatin and many significant gaps in DNA strands are not determined.

 

Conclusion

In the aspect of DNA sequencing, many methods are gradually developed from 1970s (Sanger sequencing, pyrosequencing, and so on). From those methods the Human Genome Project could be progressed in speeds which would be never possible without it. With the whole genome information in human, the personalized diagnosis will be possible and the cost will keep reduced by newer methods.

 

Reference

1. http://www.nature.com/scitable/topicpage/dna-sequencing-technologies-690

2. https://en.wikipedia.org/wiki/Sequencing#RNA_sequencing

3. http://www.genomenewsnetwork.org/resources/whats_a_genome/Chp2_2.shtml