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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

who is the first person who discover the cell
Etornam Reply
what's the difference between DNA and RNA
moffat Reply
what is a fungi
Anaba Reply
What is meant by adaptation?
Agbesi Reply
who is the father of evolution
Ferkah Reply
Charles D
Dr
complete the table below based on the levels of biological organization
Lovely Reply
Give me Examples of living thing which have 2 or more flagella?
Mahesh Reply
insect and plants
qax
bacteria and chlamydompnas
Berhanu
reproduction it's full meaning
Gift Reply
full meaning of ATP
Gifty
A life process in which living things increase their population through sexual or non sexual intercouse
Danisha
please explaination
Daniel
Gifty ATP means Adenosine tri phosphate
Mahesh
the process by which organisms produce their own kind.
Berhanu
who is the father of Biology
Ferkah
reproduction is the process where living organisms producess their offspring
jerry Reply
what is reproduction
Nmesoma Reply
why some kinds of students are failed
Ahmadi Reply
lack of concentration
Faith
lack of guidance and counseling
ali
what's the divination of openstax
John
don't mind about reading
aine
lack of focus
Afolayan
Inability to study at their own pace.📒
Agbesi
Inability to study at their own pace.📒
Agbesi
What is the meaning of optic
Kisaky Reply
Giving a specific section of the alimentary canal,describe 3 ways in which physical digestion occurs.
Kisaky
mouth when chewing
ephraim
what is population
Ivy Reply
total number of people living in an area
FILDA
a number of people lives in one catigorize area or named area
Oburak
total number of people living in a specific geographical area.📕
Agbesi
total number of people living in a specific geographical area.📕
Agbesi
what is a cell
Chiko Reply
basic and functional unit of life
Edwin
cell is tissues that makes up functional life in human or un animal.
Oburak
is the smallest basic unit of life.
Kisaky
Is the smallest baic unit. o
Kisaky
why cell is very important to human body
Ahmadi
what is diffusion
Henry
diffusion is a process of mix of particles from higher concentration to the lower one,to make the body functional normal
Adam
what is effusion
Mahesh
what is soil
FILDA Reply
Is the finely divided material covering the earth crust.
Kisaky
is the upper moist of layer of the earth surface
Ahmadi
The natural material that covers the surface of the earth crust.📖
Agbesi

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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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