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Part A shows an illustration of a DNA double helix, which has a sugar-phosphate backbone on the outside and nitrogenous base pairs on the inside. Part B shows base pairing between thymine and adenine, which form two hydrogen bonds, and between guanine and cytosine, which form three hydrogen bonds. Part C shows a molecular model of the DNA double helix. The outside of the helix alternates between wide gaps, called major grooves, and narrow gaps, called minor grooves.
DNA has (a) a double helix structure and (b) phosphodiester bonds. The (c) major and minor grooves are binding sites for DNA binding proteins during processes such as transcription (the copying of RNA from DNA) and replication.

Dna sequencing techniques

Until the 1990s, the sequencing of DNA (reading the sequence of DNA) was a relatively expensive and long process. Using radiolabeled nucleotides also compounded the problem through safety concerns. With currently available technology and automated machines, the process is cheap, safer, and can be completed in a matter of hours. Fred Sanger developed the sequencing method used for the human genome sequencing project, which is widely used today ( [link] ).

Visit this site to watch a video explaining the DNA sequence reading technique that resulted from Sanger’s work.

The method is known as the dideoxy chain termination method. The sequencing method is based on the use of chain terminators, the dideoxynucleotides (ddNTPs). The dideoxynucleotides, or ddNTPSs, differ from the deoxynucleotides by the lack of a free 3' OH group on the five-carbon sugar. If a ddNTP is added to a growing a DNA strand, the chain is not extended any further because the free 3' OH group needed to add another nucleotide is not available. By using a predetermined ratio of deoxyribonucleotides to dideoxynucleotides, it is possible to generate DNA fragments of different sizes.

Part A shows a template DNA strand and newly synthesized strands that were generated in the presence of dideoxynucleotides that terminate the chain at different points to generate fragments of different sizes. Each dideoxynucleotide is labeled a different color. Part B shows a sequence readout that was generated after the DNA fragments were separated on the basis of size. The color of the fragment indicates the identity of the nucleotide at the end of a given fragment. By reading the colors in order, the DNA sequence can be determined.
In Frederick Sanger's dideoxy chain termination method, dye-labeled dideoxynucleotides are used to generate DNA fragments that terminate at different points. The DNA is separated by capillary electrophoresis on the basis of size, and from the order of fragments formed, the DNA sequence can be read. The DNA sequence readout is shown on an electropherogram that is generated by a laser scanner.

The DNA sample to be sequenced is denatured or separated into two strands by heating it to high temperatures. The DNA is divided into four tubes in which a primer, DNA polymerase, and all four nucleotides (A, T, G, and C) are added. In addition to each of the four tubes, limited quantities of one of the four dideoxynucleotides are added to each tube respectively. The tubes are labeled as A, T, G, and C according to the ddNTP added. For detection purposes, each of the four dideoxynucleotides carries a different fluorescent label. Chain elongation continues until a fluorescent dideoxy nucleotide is incorporated, after which no further elongation takes place. After the reaction is over, electrophoresis is performed. Even a difference in length of a single base can be detected. The sequence is read from a laser scanner. For his work on DNA sequencing, Sanger received a Nobel Prize in chemistry in 1980.

Sanger’s genome sequencing has led to a race to sequence human genomes at a rapid speed and low cost, often referred to as the $1000 in one day sequence. Learn more by selecting the Sequencing at Speed animation here .

Gel electrophoresis    is a technique used to separate DNA fragments of different sizes. Usually the gel is made of a chemical called agarose. Agarose powder is added to a buffer and heated. After cooling, the gel solution is poured into a casting tray. Once the gel has solidified, the DNA is loaded on the gel and electric current is applied. The DNA has a net negative charge and moves from the negative electrode toward the positive electrode. The electric current is applied for sufficient time to let the DNA separate according to size; the smallest fragments will be farthest from the well (where the DNA was loaded), and the heavier molecular weight fragments will be closest to the well. Once the DNA is separated, the gel is stained with a DNA-specific dye for viewing it ( [link] ).

Questions & Answers

what is abiotic and biotic factors?
Hira Reply
which of the following shows the correct sequence of the cell cycle
Kameishia Reply
who is name virus
Shivam Reply
centromere consist of
Shivam
meeting point of two chromatids
Cffrrcvccgg
Explain the function of nematocysts in cnidarians?
Israel Reply
The nemotocyst is used by Cnidarians (hydra, jellyfish, sea anemones) to sting their prey and any threatening enemy.
Lee
photosynthesis in plants is an example of what ? (a) excretion (b) irritability (c) nutrition (d) reproduction
Lee Reply
If a Hox 13 gene in a mouse was replaced with a Hox 1 gene, how might this alter animal development?
Israel Reply
Which of the following organisms is most likely to be a diploblast?
Israel
what are reactions of photosynthesis?
Maria Reply
what are the probabilities of blood genotypes for the offspring from a cross between a mother lAlA blood and a father with lBi blood?
dayana Reply
what is matter
Emmanuel Reply
matter is anything that has mass and can occupied space
Alice
weight
Alice
example of matter
Oyekemi
You serves as an example of matter Because matter is anything that has mass and occupy space e.g man and every other things that exist on earth.. So think of every other things around you ...
Biola
and you too
Oyekemi
We generally
Biola
What is ecological management
Oyekemi
how the kidney functions as osmoregulatory organ
Sam Reply
That true
Banda
what is the major connection for sugars in glycolysis?
Ibrahim Reply
Simple term of science
Palesa Reply
what does it mean
gopal
it's means what do u know about biology?
Phathu
what is immunisation
Melysa
the action of making a person immune to infections ,for immunisation
Kalia
what is the biology? what do you know about biology
Phathu Reply
biology is the study of living organisms, divided into many specialized fields that cover their morphology, physiology, anatomy, behavior, origin, and distribution.
Julia
The study of all aspects of life. The study of all living organisms (such as animal cells and plant cells) in greater detail (their structure and how they function). It's a very broad science.
juanita
what is prokaryotic
Bhaskar Reply
what is pathogens
Bhaskar
pathogens are a bacterium, virus, or other microorganism that can cause disease.
Lee
transistion metals....
Wasik Reply

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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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