DNA SequencingLearning How to Read the Information Guide to Life

Soli ShinSUNY Empire Biology 1 Fall 2016

What is DNA Sequencing?We understand DNA fragments as a nucleic acid bases with sugar phosphate molecules attached to it. When these fragments are put linked together, they create the double helix that Watson & Crick announced in 1953. We will be examining how technological advances have allowed scientists to order and catalogue the information as fragments, in a process called DNA sequencing.

Currently, sequencing is carried out by machines and this presentation will focus on automated processes of DNA sequencing.

http://www2.hkedcity.net/sch_files/a/abc/abc-lnk/public_html/Biotech/dna_molecule1.html

The DNA Sequencing Machine: Early StageHow is DNA sequencing possible? Through the double helix model, we can understand that the complementing pair structure allows for one strand to inform what the complete double strand would look like. This concept is what allowed for the first automated technique -- dideoxyribonucleotide chain termination sequencing.

One strand of DNA fragment is set as a template for synthesis of a nested set of complementary fragments. These are further analyzed to yield the sequence.

http://bio1151.nicerweb.com/Locked/media/ch20/termination.html

The DNA Sequencing Machine: Next GenerationAfter dideoxy chain termination, sequencing by synthesis was adopted because it allowed to order more nucleotides in a significantly shorter period of time. The older method could order 12,000 base pairs in 10 hours; the new one could order up to 900 million base pairs in 10 hours.

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The DNA Sequencing Machine: Next GenerationSequence by synthesis works through amplifying DNA fragments that have been processed through an aqueous solution. It is copied so that the surface of a single bead of the solution can have 106 copies of the fragment. These copies are all 5’ ends and the beads are placed in wells to be matched with their 3’ ends. A solution containing one of the four nucleotides is washed over the entire plate. Each nucleotide is coded with a different light-emitting flash that indicates when it has been added to a match in the well. The fragments are then organized in a “flow-gram”.

http://www.mayomedicallaboratories.com/articles/communique/2010/05.html

The DNA Sequencing Machine: Third GenerationA single, very long DNA molecule is sequenced on its own through a nanopore. This is a very small pore, like a protein channel pore, embedded in a lipid membrane. Scientists are detecting the bases individually by the observing the effect on ions and flow of an electrical current that is being charged through the pore. Other examples of third generation sequencing include artificial membranes and nanopores.

http://www.engadget.com/2010/12/24/nanopore-dna-sequencing-technique-promises-entire-genome-in-minu/

What is Amplification?Amplifying simply means to make copies of. For example, when examining a particular trait, an investigator may amplify a targeted segment of the DNA in order to isolate which nucleotides are at causing the gene expression. Without amplification, copies of a cloned gene cannot be used in basic research or to endow another organism with a new ability. Since one gene is only a very small part of the total strand, the availability to amplify DNA fragments is crucial for any application involving a single gene.

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DNA CloningTo study specific genes, scientists have developed methods for preparing DNA segments by cloning them.

They use bacteria cells that contain plasmids. They take DNA segments and insert that segment into bacterial plasmid, which has been modified for efficient cloning. This combination of foreign DNA and bacterial plasmid is called a recombinant DNA molecule.

The plasmid is returned to a bacterial cell where it reproduces a clone of cells.

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Why Plasmids?The plasmid is an ideal cloning vector. A cloning vector indicates a DNA molecule that can carry foreign DNA into a host cell and replicate endlessly.

Readily obtained through commercial supply, plasmids from E coli cells are commonly manipulated to form recombinant plasmids. This is done in vitro. The host cells grow in a prepared culture and contains many copies of the gene of interest. A protein can then be extracted from thee genetic copies and eventually altered to make medicine or to alter gene expression to solve problems.

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Polymerase Chain ReactionWhen making copies of gene sequences, we use a technique called a polymerase chain reaction. This is a three-step cycle that produces DNA copies on an exponential scale.

Step 1: Temperature is manipulated to separate the strands and allow primers and DNA polymerase to make two pairs of complete strands.

Step 2 and 3: The process is repeated so that you have made 2 copies, then 4, then 8, etc.

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Nucleic Acid HybridizationHybridization is key to establishing and clarifying segments in DNA. Without this process, copies could not be created. The hybridization process takes the base pairing of one strand and combines it to the complementary sequence on a strand from another nucleic acid molecule. This can happen in an aqueous solution, in a gel, or even on special paper (nitrocellulose paper).

We use in situ hybridization when fluorescent dyes are applied to specific chromosomal segments that are being investigated.

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Restriction EnzymesRestriction enzymes are important components of the bacterial cell that protect it from the control sequences of foreign DNA that can launch disruptive protocols. There are many restriction enzymes and each has specific sequences of their own that correspond with a restriction site. The sites represent areas where DNA strands are cut off and can be combined with other DNA fragments, like within plasmids. Restriction enzymes are coded with 4 to 8 nucleotide pairs and we call these sequences between restriction enzymes restriction fragments.

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Sticky Ends and DNA LigaseWhen restriction enzymes cleave the sugar-phosphate backbones in the two DNA strands, the resulting double-stranded restriction fragments have what is referred to as a “sticky end”. The sticky ends are where DNA fragments from another source, such as a bacterial plasmid, can be paired with the foreign DNA.

The bonds formed are temporary until they are sealed by the enzyme DNA ligase. Ligase catalyzes the formation of covalent bonds that bind the sugar-phosphate backbones of DNA strands that were previously broken by the restriction enzymes.

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Cloning Eukaryotic GenesEukaryotic genes can be cloned despite their differences from bacterial host cells because the controls meant to order sequences get bypassed. An expression vector is used above the restriction site so that the host will identify the promoter and begin copying, even if the information is from an eukaryotic gene.

Eukaryotic cells also carry non-coding segments that bacterial cells do not. This is bypassed by using only complementary DNA by copying from mRNA which only contains exons.

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ElectroporationAnother method of introducing foreign eukaryotic DNA into a bacterial cell is through a process called electroporation. This is when a short electrical pulse runs through an aqueous solution that contains both cells. The pulse punctures small holes in the plasma membrane of the bacterial cells. Through these channels, the foreign DNA can enter.

http://www.bioelectrochemical-soc.org/img/electroporation-1.jpg

Gel ElectrophoresisIt is useful, after DNA copies have been made, to separate the bacterial plasmid from the foreign DNA. Thus, the DNA can be examined as a fragment. To study fragments of various lengths, researchers can use a process called gel electrophoresis. The gel is a polymer used as a molecular sieve to separate out a range of nucleic acids, based on their length by running an electrical charge through the gel. The shortest fragments will travel the fastest and farthest from one charged end to the other because they are lighter.

http://bio1151.nicerweb.com/Locked/media/ch20/20_08GelElectrophoresis.jpg

Gel ElectrophoresisThe agarose gel is submerged in saltwater that will conduct electricity. Slots are pre-formed in the gel and pipettes will inject the DNA into those wells. Researchers will add dye so that they can visualize the molecules as they run through the gel. DNA has a general negative change and this will make them propel to the positive charge of the other side of the gel. When UV light is shone onto the gel, the dye gives off a fluorescence that is captured on film. DNA fragments are compared to known fragment sizes.

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Evolutionary AncestryWhat can we conclude from the bacterial cell’s ability to produce proteins encoded in eukaryotic cells? We can look backwards at our course timeline to when we answered a similar question about how these two type of cells originated. We can theorize that because of their shared mechanism of gene expression, they had a common ancestor, through which, we can still witness evolutionary roots. This also means that given the right conditions, genes that are taken from one species can function and be expressed in an entirely different species.

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The Human Genome ProjectThe Human Genome Project is a government- led initiative to map the entire sequence of the human genome. The main and most immediate benefits of mapping the human genome was the information available to medical professionals who would be able to interpret the data. Research has been on the rise about diseases that are inherited genetically. If there is an identifiable segment of DNA that links with a disease, then there may be a way to prevent these ailments from occurring. This project has led to offshoots such as the Cancer Genome Project, which will be pivotal to cancer-related trials, drugs, and prevention.

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Genetic EngineeringThis presentation brings all of the tools of DNA sequencing into further detail and concludes with a question outside the realm of pure scientific inquiry: the issue of direct manipulation of genes for practical purposes. Regardless of how we as a species use genetic engineering, no one can deny the groundbreaking medicinal achievements that have been made due to our ability to sequence and order DNA. Ultimately, our descendants will decide how we use future technology but for now, we can only hope that we will obtain progress in how we battle genetic diseases.

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References:https://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

https://www.genome.gov/10001177

● http://www.genome.gov/10001772○ http://report.nih.gov/NIHfactsheets/ViewFactSheet.aspx?csid=45&key=H#H○ http://www.genome.gov/11006943

https://www.dnalc.org/resources/animations/sangerseq.html

● https://www.dnalc.org/resources/animations/gelelectrophoresis.html

Pearson Textbook: Campbell Biology Tenth Edition