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The left part of this illustration shows a parent strand of DNA with the sequence GATTCAGC, and four daughter strands, each of which was made in the presence of a different dideoxynucleotide: ddATP, ddCTP, ddGTP, or ddTTP. The growing chain terminates when a ddNTP is incorporated, resulting in daughter strands of different lengths. The right part of this image shows the separation of the DNA fragments on the basis of size. Each ddNTP is fluorescently labeled with a different color so that the sequence can be read by the size of each fragment and its color.
Frederick Sanger's dideoxy chain termination method is illustrated. Using dideoxynucleotides, the DNA fragment can be terminated at different points. The DNA is separated on the basis of size, and these bands, based on the size of the fragments, can be read.

Early strategies: shotgun sequencing and pair-wise end sequencing

In shotgun sequencing    method, several copies of a DNA fragment are cut randomly into many smaller pieces (somewhat like what happens to a round shot cartridge when fired from a shotgun). All of the segments are then sequenced using the chain-sequencing method. Then, with the help of a computer, the fragments are analyzed to see where their sequences overlap. By matching up overlapping sequences at the end of each fragment, the entire DNA sequence can be reformed. A larger sequence that is assembled from overlapping shorter sequences is called a contig    . As an analogy, consider that someone has four copies of a landscape photograph that you have never seen before and know nothing about how it should appear. The person then rips up each photograph with their hands, so that different size pieces are present from each copy. The person then mixes all of the pieces together and asks you to reconstruct the photograph. In one of the smaller pieces you see a mountain. In a larger piece, you see that the same mountain is behind a lake. A third fragment shows only the lake, but it reveals that there is a cabin on the shore of the lake. Therefore, from looking at the overlapping information in these three fragments, you know that the picture contains a mountain behind a lake that has a cabin on its shore. This is the principle behind reconstructing entire DNA sequences using shotgun sequencing.

Originally, shotgun sequencing only analyzed one end of each fragment for overlaps. This was sufficient for sequencing small genomes. However, the desire to sequence larger genomes, such as that of a human, led to the development of double-barrel shotgun sequencing, more formally known as pairwise-end sequencing . In pairwise-end sequencing, both ends of each fragment are analyzed for overlap. Pairwise-end sequencing is, therefore, more cumbersome than shotgun sequencing, but it is easier to reconstruct the sequence because there is more available information.

Next-generation sequencing

Since 2005, automated sequencing techniques used by laboratories are under the umbrella of next-generation sequencing    , which is a group of automated techniques used for rapid DNA sequencing. These automated low-cost sequencers can generate sequences of hundreds of thousands or millions of short fragments (25 to 500 base pairs) in the span of one day. These sequencers use sophisticated software to get through the cumbersome process of putting all the fragments in order.

Evolution connection

Comparing sequences

A sequence alignment is an arrangement of proteins, DNA, or RNA; it is used to identify regions of similarity between cell types or species, which may indicate conservation of function or structures. Sequence alignments may be used to construct phylogenetic trees. The following website uses a software program called BLAST (basic local alignment search tool) .

Under “Basic Blast,” click “Nucleotide Blast.” Input the following sequence into the large "query sequence" box: ATTGCTTCGATTGCA. Below the box, locate the "Species" field and type "human" or "Homo sapiens". Then click “BLAST” to compare the inputted sequence against known sequences of the human genome. The result is that this sequence occurs in over a hundred places in the human genome. Scroll down below the graphic with the horizontal bars and you will see short description of each of the matching hits. Pick one of the hits near the top of the list and click on "Graphics". This will bring you to a page that shows where the sequence is found within the entire human genome. You can move the slider that looks like a green flag back and forth to view the sequences immediately around the selected gene. You can then return to your selected sequence by clicking the "ATG" button.

Use of whole-genome sequences of model organisms

The first genome to be completely sequenced was of a bacterial virus, the bacteriophage fx174 (5368 base pairs); this was accomplished by Fred Sanger using shotgun sequencing. Several other organelle and viral genomes were later sequenced. The first organism whose genome was sequenced was the bacterium Haemophilus influenzae ; this was accomplished by Craig Venter in the 1980s. Approximately 74 different laboratories collaborated on the sequencing of the genome of the yeast Saccharomyces cerevisiae , which began in 1989 and was completed in 1996, because it was 60 times bigger than any other genome that had been sequenced. By 1997, the genome sequences of two important model organisms were available: the bacterium Escherichia coli K12 and the yeast Saccharomyces cerevisiae . Genomes of other model organisms, such as the mouse Mus musculus , the fruit fly Drosophila melanogaster , the nematode Caenorhabditis. elegans , and humans Homo sapiens are now known. A lot of basic research is performed in model organisms because the information can be applied to genetically similar organisms. A model organism    is a species that is studied as a model to understand the biological processes in other species represented by the model organism. Having entire genomes sequenced helps with the research efforts in these model organisms. The process of attaching biological information to gene sequences is called genome annotation    . Annotation of gene sequences helps with basic experiments in molecular biology, such as designing PCR primers and RNA targets.

Click through each step of genome sequencing at this site .

Uses of genome sequences

DNA microarrays are methods used to detect gene expression by analyzing an array of DNA fragments that are fixed to a glass slide or a silicon chip to identify active genes and identify sequences. Almost one million genotypic abnormalities can be discovered using microarrays, whereas whole-genome sequencing can provide information about all six billion base pairs in the human genome. Although the study of medical applications of genome sequencing is interesting, this discipline tends to dwell on abnormal gene function. Knowledge of the entire genome will allow future onset diseases and other genetic disorders to be discovered early, which will allow for more informed decisions to be made about lifestyle, medication, and having children. Genomics is still in its infancy, although someday it may become routine to use whole-genome sequencing to screen every newborn to detect genetic abnormalities.

In addition to disease and medicine, genomics can contribute to the development of novel enzymes that convert biomass to biofuel, which results in higher crop and fuel production, and lower cost to the consumer. This knowledge should allow better methods of control over the microbes that are used in the production of biofuels. Genomics could also improve the methods used to monitor the impact of pollutants on ecosystems and help clean up environmental contaminants. Genomics has allowed for the development of agrochemicals and pharmaceuticals that could benefit medical science and agriculture.

It sounds great to have all the knowledge we can get from whole-genome sequencing; however, humans have a responsibility to use this knowledge wisely. Otherwise, it could be easy to misuse the power of such knowledge, leading to discrimination based on a person's genetics, human genetic engineering, and other ethical concerns. This information could also lead to legal issues regarding health and privacy.

Section summary

Whole-genome sequencing is the latest available resource to treat genetic diseases. Some doctors are using whole-genome sequencing to save lives. Genomics has many industrial applications including biofuel development, agriculture, pharmaceuticals, and pollution control. The basic principle of all modern-day sequencing strategies involves the chain termination method of sequencing.

Although the human genome sequences provide key insights to medical professionals, researchers use whole-genome sequences of model organisms to better understand the genome of the species. Automation and the decreased cost of whole-genome sequencing may lead to personalized medicine in the future.

Questions & Answers

what is used to determine phylogeny?
Israel Reply
which condition is the basis for a species to be reproductively isolated from other members?
Israel Reply
Why do scientists consider vestigial structures evidence for evolution?
Israel
8.Which statement about analogies is correct?
Israel
What is true about organisms that are a part of the same clade?
Israel
Why is it so important for scientists to distinguish between homologous and analogous characteristics before building phylogenetic trees?
Israel
(CH2O)n is the stoichiometric formula of
Marcellus Reply
what are nucleotide
Anastijjaninaiya Reply
Methane,ammonia,water and sugar are dissolved to form nuceotide
Me
Introduction To Biology
Tanveer Reply
can ringworm be caused by bacterium
fred Reply
Nope
ejikeme
Branches of biology
Tanveer
no it does not occurs by bacterium
gopal
what is a brick?
Istifanus Reply
what is gene in biology?
yousaf Reply
it is a heredity unit
Me
what is DNA
yousaf Reply
carrier of genetic information
missy
deoxyribonucleic acid
gopal
it contains genetic information and brings it to one generation to other
gopal
it is of two Types circular DNA and linear DNA
gopal
plasmids are the type of small circular DNA which lies outside the genomic DNA
gopal
And what makes a virus to be difficult to destroy
Mosongo
what observation is made when dry seeds and soaked seeds are put in a vacuum flask
Robin Reply
there is respiration from the soak seeds which shows on the walls of the vacuum flask
Israel
what's mammals ?
Istifanus Reply
mammals are vertebrates ,any member group of vertebrates animals in which the young are nourished with milk from special mammary glands of the mother.
yousaf
what are actin and myosin
Praveen Reply
they are muscle filaments
Israel
they make up the microfibrils of the muscle ,relaxing and contracting to cause movement
Israel
Please did anybody know the questions that will come out in the coming practical?
Oladimeji
Why it is importantthat there are different types of protein in plasma membraine for the transport materials into and out of a cell?
Louellie Reply
the nerve cell
Mustapha Reply
differences between Homo sapiens and other primates
Aphiwe Reply
Why is albinism a recessive trait
Bright
Tyrosinase gene are make albinism to recessive trait
vinod
And what are the Tyrosinase genes
Bright

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Source:  OpenStax, Biology. OpenStax CNX. Feb 29, 2016 Download for free at http://cnx.org/content/col11448/1.10
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