Purification of circular YACs from yeast cells for DNA sequencing

We describe a method for the purification of circular yeast artificial chromosome (YAC) DNA 120–150 kilobases (kb) in size that is of sufficient quantity and quality for restriction enzyme analysis and DNA sequencing. This method preferentially enriches for circular YAC DNA and avoids the time-consuming step of centrifugation in CsCl – ethidium bromide (EtBr) gradients. We applied this method to the purification of circular YACs carrying DNA segments that are extremely unstable in E. coli, including those that correspond to GAP2 and GAP3 on human chromosome 19. We showed that YAC DNA (GAP2 and GAP3) purified using this new method is clearly resolved in EtBr-stained gels. The sequence of YAC-GAP3 was obtained, representing the first GAP clone sequenced in YAC form. At present, it is estimated that there are more than 1000 gaps in the human genome that cannot be cloned using bacterial vectors. Thus, our new method may be very useful for completing the last stage of the human genome project.

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2. How to read the book of life

Genomics: A Very Short Introduction ◽

10.1093/actrade/9780198786207.003.0002 ◽

2018 ◽

pp. 12-27

Author(s):

John Archibald

Keyword(s):

Dna Sequencing ◽

Human Genome ◽

Single Molecule ◽

Human Genome Project ◽

Genome Project ◽

Chain Termination ◽

Single Molecule Sequencing ◽

Semiconductor Sequencing ◽

The Cost ◽

The Human Genome Project

For all its biological importance, DNA is a fragile molecule so extracting it is a difficult process. ‘How to read the book of life’ explains the techniques required to sequence DNA. It begins by explaining the techniques developed for protein and RNA sequencing by Frederick Sanger, Robert Holley, and Carl Woese that were then developed further for DNA sequencing. Following the success of the Human Genome Project, the next generation of DNA sequencing was developed in the mid-2000s. Pyrosequencing was capable of generating orders of magnitude more data at a fraction of the cost, but was superceded within a decade by semiconductor sequencing, reversible chain-termination sequencing, and single-molecule sequencing.

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Recombinant DNA technology and DNA sequencing

Essays in Biochemistry ◽

10.1042/ebc20180039 ◽

2019 ◽

Vol 63 (4) ◽

pp. 457-468 ◽

Cited By ~ 1

Author(s):

Mark A. J. Roberts

Keyword(s):

Dna Sequencing ◽

Human Genome ◽

Dna Sequences ◽

Human Genome Project ◽

Recombinant Dna ◽

Genome Project ◽

Recombinant Dna Technology ◽

Dna Technology ◽

The Human Genome Project

Abstract DNA present in all our cells acts as a template by which cells are built. The human genome project, reading the code of the DNA within our cells, completed in 2003, is undoubtedly one of the great achievements of modern bioscience. Our ability to achieve this and to further understand and manipulate DNA has been tightly linked to our understanding of the bacterial and viral world. Outside of the science, the ability to understand and manipulate this code has far-reaching implications for society. In this article, we explore some of the basic techniques that enable us to read, copy and manipulate DNA sequences alongside a brief consideration of some of the implications for society.

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DNA Sequencing and the Human Genome Project

Molecular Biology in Cellular Pathology ◽

10.1002/0470867949.ch15 ◽

2003 ◽

pp. 307-328

Author(s):

Philip Bennett

Keyword(s):

Dna Sequencing ◽

Human Genome ◽

Human Genome Project ◽

Genome Project ◽

The Human Genome Project

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Cultivating DNA Sequencing Technology After the Human Genome Project

Annual Review of Genomics and Human Genetics ◽

10.1146/annurev-genom-111919-082433 ◽

2020 ◽

Vol 21 (1) ◽

pp. 117-138

Author(s):

Jeffery A. Schloss ◽

Richard A. Gibbs ◽

Vinod B. Makhijani ◽

Andre Marziali

Keyword(s):

Dna Sequencing ◽

Human Genome ◽

Human Genome Project ◽

Technology Development ◽

Genome Project ◽

Alternative Methods ◽

Biological Research ◽

Sequencing Technology ◽

Core Technology ◽

The Human Genome Project

When the Human Genome Project was completed in 2003, automated Sanger DNA sequencing with fluorescent dye labels was the dominant technology. Several nascent alternative methods based on older ideas that had not been fully developed were the focus of technical researchers and companies. Funding agencies recognized the dynamic nature of technology development and that, beyond the Human Genome Project, there were growing opportunities to deploy DNA sequencing in biological research. Consequently, the National Human Genome Research Institute of the National Institutes of Health created a program—widely known as the Advanced Sequencing Technology Program—that stimulated all stages of development of new DNA sequencing methods, from innovation to advanced manufacturing and production testing, with the goal of reducing the cost of sequencing a human genome first to $100,000 and then to $1,000. The events of this period provide a powerful example of how judicious funding of academic and commercial partners can rapidly advance core technology developments that lead to profound advances across the scientific landscape.

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