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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

fabace family plant name
Pushpam Reply
in eukaryotes ...protein channel name which transport protein ...
Pushpam Reply
in bacteria ...chromosomal dna duplicate structure called
what is a prokaryotic cell and a eukaryotic cell
Matilda Reply
There are two types of cells. Eukaryotic and Prokaryotic cells. Prokaryotic cells don't have a nucleus or membrane enclosed organelles (little organs within that cell). They do however carry genetic material but it's not maintained in the nucleus. Prokaryotic cells are also one celled.
Prokaryotic cells are one celled (single celled).
Prokaryotic cells are Bacteria and Archea
Prokaryotic cells are smaller than Eukaryotic cells.
Eukaryotic cells are more complex. They are much bigger than Prokaryotic cells.
Eukaryotic cells have a nucleus and membrane bound organelles.
Eukaryotic cells are animals cells which also includes us.
Eukaryotic cells are also multicellular.
nice explaination
eukaryotic cells are individual cells .. but eukaryotes are multicellular organisms which consist of many different types of eukaryotic cells
also eukaryotic cells have mitochondria. prokaryotic cells do not
in prokaryotes only ribosomes are present... in eukaryotes mitochondria ...glogi bodies ..epidermis .....prokaryotes one envelop but eukaryotes compartment envelop....envelop mean membrane bound organelles......
we have 46 pair of somatic cell and 23 pair of chromosomes in our body, pls can someone explain it to me. pls
Matilda Reply
we have 22 pairs of somatic chromosomes and one pair of sex chromosome
we have 23 pairs of chromosomes,22 pairs of somatic and one pair of sex chromosomes
23 chromosomes from dad & 23 chromosomes from mom 23 +23=46 total chromosomes
X & Y chromosomes are called sex cells, the very presence of a Y chromosome means the person is Male.
XX Female XY Male
If a Karyotype has more than 46 Chromosomes then nondisjunction occured. For example, having an extra chromosome 21 will cause Down Syndrome.
in mammal state the different vertebrae and their location in the body
Igbinigie Reply
what is a somatic cell
Senam Reply
somatic cells are body cells
somatic cell organised whole plant body part
what is biology
lilian Reply
what is biology
Dada Reply
The scientific study of life.
the virus that causes mumps in humans is composed of a protein outer Shell containing a core of DNA
Daniel Reply
Basic science and applied science question about cancer
Joyce Reply
what are the importance of ATPs
Olatunji Reply
How can biology be studied from a microscopic approach to a global approach
Joyce Reply
The large central opening in the poriferan body is called
Chynna Reply
You go for a long walk on a hot day. Give an example of a way in which homeostasis keeps your body healthy.
Joyce Reply
You sweat.
sweating is your bodies way of keeping you from overheating.
Thank you
what is biology
Neya Reply
biology is the study of life
Biology is the study of Life
is the branch of science which deals with the of living things.

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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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