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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

how can chip be made from sand
Eke Reply
is this allso about nanoscale material
Almas
are nano particles real
Missy Reply
yeah
Joseph
Hello, if I study Physics teacher in bachelor, can I study Nanotechnology in master?
Lale Reply
no can't
Lohitha
where is the latest information on a no technology how can I find it
William
currently
William
where we get a research paper on Nano chemistry....?
Maira Reply
nanopartical of organic/inorganic / physical chemistry , pdf / thesis / review
Ali
what are the products of Nano chemistry?
Maira Reply
There are lots of products of nano chemistry... Like nano coatings.....carbon fiber.. And lots of others..
learn
Even nanotechnology is pretty much all about chemistry... Its the chemistry on quantum or atomic level
learn
Google
da
no nanotechnology is also a part of physics and maths it requires angle formulas and some pressure regarding concepts
Bhagvanji
hey
Giriraj
Preparation and Applications of Nanomaterial for Drug Delivery
Hafiz Reply
revolt
da
Application of nanotechnology in medicine
has a lot of application modern world
Kamaluddeen
yes
narayan
what is variations in raman spectra for nanomaterials
Jyoti Reply
ya I also want to know the raman spectra
Bhagvanji
I only see partial conversation and what's the question here!
Crow Reply
what about nanotechnology for water purification
RAW Reply
please someone correct me if I'm wrong but I think one can use nanoparticles, specially silver nanoparticles for water treatment.
Damian
yes that's correct
Professor
I think
Professor
Nasa has use it in the 60's, copper as water purification in the moon travel.
Alexandre
nanocopper obvius
Alexandre
what is the stm
Brian Reply
is there industrial application of fullrenes. What is the method to prepare fullrene on large scale.?
Rafiq
industrial application...? mmm I think on the medical side as drug carrier, but you should go deeper on your research, I may be wrong
Damian
How we are making nano material?
LITNING Reply
what is a peer
LITNING Reply
What is meant by 'nano scale'?
LITNING Reply
What is STMs full form?
LITNING
scanning tunneling microscope
Sahil
how nano science is used for hydrophobicity
Santosh
Do u think that Graphene and Fullrene fiber can be used to make Air Plane body structure the lightest and strongest. Rafiq
Rafiq
what is differents between GO and RGO?
Mahi
what is simplest way to understand the applications of nano robots used to detect the cancer affected cell of human body.? How this robot is carried to required site of body cell.? what will be the carrier material and how can be detected that correct delivery of drug is done Rafiq
Rafiq
if virus is killing to make ARTIFICIAL DNA OF GRAPHENE FOR KILLED THE VIRUS .THIS IS OUR ASSUMPTION
Anam
analytical skills graphene is prepared to kill any type viruses .
Anam
Any one who tell me about Preparation and application of Nanomaterial for drug Delivery
Hafiz
what is Nano technology ?
Bob Reply
write examples of Nano molecule?
Bob
The nanotechnology is as new science, to scale nanometric
brayan
nanotechnology is the study, desing, synthesis, manipulation and application of materials and functional systems through control of matter at nanoscale
Damian
how did you get the value of 2000N.What calculations are needed to arrive at it
Smarajit Reply
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what does post-translational control refer to?
Teresa Reply
Bioremediation includes
Rachel Reply

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Source:  OpenStax, Ucd bis2a intro to biology v1.2. OpenStax CNX. Sep 22, 2015 Download for free at https://legacy.cnx.org/content/col11890/1.1
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