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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?
Williams Reply
state 2 abiotic factors that affect the rate of transpiration in plants?
Benenge
non living things
Chris
what is a cell
Esther Reply
what is contained in a smooth endoplasmic reticulum?
Bleyd Reply
sperm
Chris
cool
Jo
sorry i meant the structure within the smooth endoplasmic reticulum
Bleyd
what is cell made of
Jo Reply
what is sponge
Joseph Reply
what is a photometer
Abigail Reply
what is respiration
Alao Reply
Is the release of energy from food substance in a living organism
innocent
What is cytoplasm
Nitta Reply
Parasitic adaptation of tapeworm
Angela Reply
Describe osmoregulator or osmoconformers and how these tools allow animals to adapt to different environment.
Alick Reply
describe the secondary function of a leaf to a plant.
Twizera Reply
hi I'm asking a question about HIV infection, can HIV infection transmitted from Mother to unbron child? please help me I So confused.
Khushboy Reply
no it can't
Ahmad
yes it can but their is an injection that can be used to prevent it.
Gift
Ahmad how no?
Khushboy
Hi im new
innocent
no, unless when giving birth
Bright
YES
Kaole
it is can be transmitted but there is an injection that the mother is injected to prevent the disease
Nia
OK
Kaole
I agree with nia
peter
but I think HIV infection can be transmitted through blood so the unborn baby can be affected by HIV when a slight mistake occurred that what i think
Salim
yes, but there is a treatment however, using a retroviral therapy and injection to prevent the unborn child unscathed from the infection
Gula
what is the difference between primary and secondary active transport in detail? I didn't understand the steps in the textbook specifically
Fathima Reply
you are a doctor?
Mohammed
what is the meaning of connective tissue?
Mohammed Reply
what are the characteristics of living things
Owolo Reply
hi
Mohammed
what's the meaning of connective tissue?
Mohammed
Reproduction, adaptation, interaction, movement, growth, respiration, made of cells, responsive to environment (homeostasis), metabolic action (consumption of food converted into energy)
garret
Movement, reproduction, nutrition, irritability, growth, excretion, respiration, death, adaptation, competition
peter

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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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