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Genetic Sequencing: Past, Present, and Future

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Naiomi Gunaratne

on 14 November 2012

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Transcript of Genetic Sequencing: Past, Present, and Future

Leighann Panico, Reshma Muppala,
Naiomi Gunaratne, Brett Riederman Genetic Sequencing:
Past, Present, and Future What is
Genetic Sequencing? The Past The Present The Future
and Implications Genome sequencing is the process of determining the order of DNA nucleotides in a genome. In other terms, the order of As, Cs, Gs, and Ts that make up the DNA of an organism.
The introduction of fast DNA sequencing methods has greatly enhanced the growth biological and medical research and discovery. What is the purpose of this? Sequencing the genome is an important step towards understanding it.
Scientists believe that being able to study the entire genome sequence will help them understand how the genes of the genome interact to guide the growth and development of the organism. How do you sequence a genome? Scientists sequence a genome in pieces, and then reassemble them to analyze the entire genome. There are two approaches to this: Clone-by-clone method Involves breaking the genome up into “clones” of approximately 150,000 bp. First the clones are ordered on a map. This compartmentalization reduces the complexity due to repetitive sequences. Then each clone is cut into a smaller, overlapping pieces of 500 bp each to sequence. The smaller pieces are sequenced and the overlaps are used to reconstruct the entire genome. "Whole-genome shotgun” sequencing This involves breaking the genome up into small fragments, sequencing the pieces at random, and reassembling the pieces by looking for overlaps back into the full genome sequence. This method works well for bacterial genomes, which are small. Pros and Cons of the Two Methods Each of these approaches has advantages and disadvantages. The clone-by-clone method is reliable but slow, and the mapping step can be especially long. By contrast, the whole-genome shotgun method is potentially very quick, but it can be extremely tricky to put together so many little pieces of sequence all at once. 1953: Watson and Crick propose the double helix shape of a DNA molecule
1970: Type II restriction enzymes discovered -> instrumental in the creation of genome sequencing methods
1972: Development of recombinant DNA
1977: Maxam-Gilbert sequencing -> DNA chemical modification
1977: Frederick Sanger develops his “Sanger Method” (Dideoxy Method) of sequencing DNA -> uses chain-terminating inhibitors; became the more efficient method 1977: Bacteriophage Phi X 174 is the first DNA genome to be fully sequenced
1986: Leroy Hood at the California Institute of Technology creates a semi-automatic DNA sequencing machine -> altered Sanger’s method by using fluorescent dyes in place of radioisotopes
1995: The shotgun sequencing methods is developed in order to sequence long strands of DNA -> Strands are cut, short sequences are determined using Sanger method, and DNA is pieced together by means of computer programming
1995: Craig Venter and Hamilton Smith publish the first sequenced genome of a living organism: Haemophilus Influenzae 1996 – Present: “Next Generation” Sequencing Methods
Pyrosequencing: Analyzes intensities of light emitted after a new nucleotide is added to a DNA sequence. Incorporation of a nucleotide releases pyrophosphate.
Massively Parallel Signature Sequencing (MPSS): small nucleotide sequences from mRNA are cloned and attached to microbeads. Sequences are then identified by their attachment to fluorescent encoder strands. Produced by Oxford Nanopore
Cost: $900
Can sequence 100 million base pairs within 6 hours with provided reagents
Important because: can analyze microRNAs, which can be a very early indicator of diseases, including sepsis Nanopore Technology: Nanopores 1. Voltage difference between two halves of fluid

2. Current “sweeps” molecules of DNA, proteins, RNA into mouth of nanopore

3. Recall, target molecule will lodge into nanopore so that potential energy is lowest Nanopore Technology: Strand Sequencing 1. DNA attracted to enzyme

2. Enzyme attracted to nanopore

3. Enzyme unzips double helix

4. DNA strand moves one base at a time through nanopore

5. Sensors detect differentiated currents depending on which base goes through nanopore Fetal Sequencing Produced by: University of Washington
Cost: $50,000
Mom’s blood + dad’s spit = entire expected fetal genome
Taken at 8 weeks
Highlights mutations
Double checking with umbilical cord blood after baby was born showed >98% accuracy Ethical Implications Genetic Counseling
Is it beneficial to know the genome of an unborn human?
Yes
Can this information be misused?
Yes, may result in genetically engineered babies or result in “positive selection” New Fields Predictive and personalized medicine
Involves predicting the probablity of a disease and instituting preventitive measures
DNA sequencing give a more definitive picture than CAT scans, MRI’s, amniocentesis, etc. The Economy DNA sequencing can result in an overall lower cost for health care
Sequencing has gone from millions of dollars to $6000 for a full human genome
Need to balance the cost of sequencing with the cost of remedial treatment Focus During the 2000s: Genome Sequencing Not focused on the methods, but using the methods
Sequencing the whole genome of organisms
Mice
Rats
Humans The Human Genome Project US Department of Energy and NIH
Craig Venter
Started in 1990, completed in 2003
Still papers published on accuracy and even more completion of the genome
2006 - last 24 chromosomes
Using the shotgun approach
Huge milestone - opened the door to even more research 2010s: Utilizing Genetic Sequencing More Often Genetic sequencing of MRSA afflicted babies in British Hospital
Noticed common sequences - able to see that one outbreak
Trace to carriers
Treated to prevent further outbreaks
"We think this is the first case where whole genome sequencing has actually led to a clinical intervention and brought the outbreak to a close."
Cheap to administer New Focus: New Techniques Personalized medicine
Less costly methods
More efficient and quick THE END
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