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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

Is the "growth and maintenance phase" in a cell's life cycle when cell division is about to occur
Somto Reply
what is the common name of Basidiomycetes
Ogechukwu Reply
الاجزاء النباتية لابد من تعقيمها قبل زراعتها في القوارير
yes
tariq
whats this?
tariq
do you speak arabic?!
what are bio elements
Shahzad Reply
which are present In Body And such elements Have Great role in our Body there are 16 bio elements that maintains human Body but on The basis of amount There are 6 bio elements present in Concen. of 99% and More Valuable And Highly Concen. element is Oxygent with 65 %
Haider
how je pollution brought about
Lamina Reply
how je pollution brouhgt about
Lamina
non is pollution brouhgt about
Lamina
describe the anatomy of cell division
Ivanovic Reply
Complex traits such as height result from 
Ruben Reply
what is the difference between chloroplasts and mitochondria
Nkalubo Reply
chloroplast in plants and bacterial cell ; mitochondria in animal cells
aung
Diagram of a living cell
Eliza Reply
what is cell
Sule
A cell is the smallest basic unit of life.
John
what's biology
Ogochukwu Reply
this is da study of living and non-living thing in an eco-system
Nutty
it is the study of living and non living organism in the ecology
Akufia
I agree with you dat biology is d study of living nd nonliving features
Winner
why do plants store carbohydrates in form of starch and not glucose?
Nutty Reply
Describe the structure of starch?
Nutty
wat is diffusion
Winner
water is life!.. Discuss?
Nutty Reply
why do plants store carbohydrates in form if starch not glucose!
Nutty
study of living thing
Dennis Reply
what is beyond a liveing cell
Raymond
what is biology
Gabriel Reply
d study of living nd non living thing
Winner
what is vasectomy
Evelyn Reply
The surgical removal of d spermduct
Eniola

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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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