Only the pairing between a purine and pyrimidine can explain the uniform diameter. That is to say, at each point along the DNA molecule, the two sugar phosphate backbones are always separated by three rings, two from a purine and one from a pyrimidine. The two strands are held together by base pairing between nitrogenous bases of one strand and nitrogenous bases from the other strand.
Base pairing takes place between a purine and pyrimidine stabilized by hydrogen bonds: A pairs with T via two hydrogen bonds and G pairs with C via three hydrogen bonds. The interior basepairs rotate with respect to one another, but are also stacked on top of each other when the molecule is viewed looking up or down its long axis.
Each base pair is separated from the previous base pair by a height of 0. Therefore, ten base pairs are present per turn of the helix. Rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.
Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous applied fields such as diagnostics, biotechnology, forensic biology, and biological systematics. The rapid speed of sequencing attained with modern technology has been instrumental in obtaining complete DNA sequences, or genomes, of numerous types and species of life, including the human genome and those of other animal, plant, and microbial species.
However, until the s, the sequencing 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 cheaper, safer, and can be completed in a matter of hours. The Sanger sequencing method was used for the human genome sequencing project, which was finished its sequencing phase in , but today both it and the Gilbert method have been largely replaced by better methods.
The DNA is separated by capillary electrophoresis on the basis of size. From the order of fragments formed, the DNA sequence can be read. The smallest fragments were terminated earliest, and they come out of the column first, so the order in which different fluorescent tags exit the column is also the sequence of the strand.
The DNA sequence readout is shown on an electropherogram that is generated by a laser scanner. The Sanger method is also known as the dideoxy chain termination method. This sequencing method is based on the use of chain terminators, the dideoxynucleotides ddNTPs. By using a predetermined ratio of deoxyribonucleotides to dideoxynucleotides, it is possible to generate DNA fragments of different sizes when replicating DNA in vitro.
A Sanger sequencing reaction is just a modified in vitro DNA replication reaction. The ddNTPs are what distinguish a Sanger sequencing reaction from just a replication reaction. But at random locations, it will instead add a ddNTP. When it does, that strand will be terminated at the ddNTP just added. If enough template DNAs are included in the reaction mix, each one will have the ddNTP inserted at a different random location, and there will be at least one DNA terminated at each different nucleotide along its length for as long as the in vitro reaction can take place about nucleotides under optimal conditions.
The ddNTPs which terminate the strands have fluorescent labels covalently attached to them. After the reaction is over, the reaction is subject to capillary electrophoresis. All the newly synthesized fragments, each terminated at a different nucleotide and so each a different length, are separated by size. As each differently-sized fragment exits the capillary column, a laser excites the flourescent tag on its terminal nucleotide. From the color of the resulting flouresence, a computer can keep track of which nucleotide was present as the terminating nucleotide.
The four reaction products were then separated by gel electrophoresis, a process that organizes DNA fragments in order of size. This enabled researchers to assess the lengths of the truncated strands in each sample.
This was important, because the end of each truncated strand was used to determine the position at which a ddNTP was added to the strand, thereby halting DNA elongation. This page appears in the following eBook. Aa Aa Aa. How do researchers "read" gene sequences? Determining the order of the nucleotides within a gene is known as DNA sequencing.
The earliest DNA sequencing methods were time consuming, but a major breakthrough came in with the development of the process called Sanger sequencing. Sanger sequencing is named after English biochemist Frederick Sanger, and it is sometimes also referred to as chain-termination sequencing or dideoxy sequencing. Some 25 years after its creation, the Sanger method was used to sequence the human genome, and, with the addition of many technological improvements and modifications, it remains an important method in laboratories across the world today.
How does Sanger sequencing work? Understanding DNA replication. Setting up the sequencing experiment. Adding ddNTPs. Figure 2: The four ddNTPs. Figure 3: By adding together information about all of the truncated strands, researchers can determine the nucleotide sequence of the DNA target. The sugar-phosphate backbone is depicted as gray, horizontal cylinders stacked end-to-end.
Each cylinder is attached to a thin rectangle, representing the nucleotide. Gray nucleotides have an unknown chemical composition. Green nucleotides represent adenine, and orange nucleotides represent cytosine. The sequence of nucleotides is: two gray, green, orange, gray, orange, two gray, green, 5 gray, green, gray.
In the bottom DNA strand, eight nucleotides are base paired with the upper strand on the right side. The second sugar-phosphate group is colored black instead of gray, indicating that it contains a dideoxy-ribose sugar, and the first nucleotide is off-set to indicate that it is not bound to the DNA chain. The sequence of the paired nucleotides is: red thymine , blue guanine , orange, blue, green, orange, red, blue. In a smaller diagram to the left of the larger chain, examples of resulting truncated nucleotide chains help decipher the DNA sequence.
Under the heading ddTTP, three nucleotide chains are shown. The first chain contains 14 nucleotides, with a red ddTTP inserted in the left-most position, truncating synthesis. The second chain contains 8 nucleotides, also truncated with a ddTTP. The third chain contains only 2 nucleotides, truncated after ddTTP addition. Under the heading ddGTP, two nucleotide chains are shown. The first chain contains 13 nucleotides, truncated after ddGTP addition.
The second chain contains 11 nucleotides, also truncated after ddGTP addition. After complete analysis with all four ddNTPs, the final nucleotide sequence is shown in the right panel. This mRNA molecule carries DNA's message from the nucleus to ribosomes in the cytoplasm, where proteins are assembled. However, before it can do this, the mRNA strand must separate itself from the DNA template and, in some cases, it must also undergo an editing process of sort.
In this view, the 5' end of the RNA strand is in the foreground. Note the inclusion of uracil yellow in RNA. Termination and editing. Figure 6: In eukaryotes, noncoding regions called introns are often removed from newly synthesized mRNA.
One extends from the upper left corner to the mid-right side. The other strand forms a loop, with the two ends pinched together and nearly touching the first strand. The sugar-phosphate backbone is depicted as a segmented white cylinder.
Nitrogenous bases are represented as blue, green, yellow, or red vertical rectangles extending downward from each segment on the sugar-phosphate backbone. The loop represents a section of mRNA, called an intron, that has been removed from the coding sequence. This process is referred to as termination. In eukaryotes, the process of termination can occur in several different ways, depending on the exact type of polymerase used during transcription.
In some cases, termination occurs as soon as the polymerase reaches a specific series of nucleotides along the DNA template, known as the termination sequence. In other cases, the presence of a special protein known as a termination factor is also required for termination to occur.
Figure 7: In eukaryotes, a poly-A tail is often added to the completed, edited mRNA molecule to signal that this molecule is ready to leave the nucleus through a nuclear pore. At this point, at least in eukaryotes, the newly synthesized mRNA undergoes a process in which noncoding nucleotide sequences, called introns , are clipped out of the mRNA strand. This process "tidies up" the molecule and removes nucleotides that are not involved in protein production Figure 6.
Then, a sequence of adenine nucleotides called a poly-A tail is added to the 3' end of the mRNA molecule Figure 7. This sequence signals to the cell that the mRNA molecule is ready to leave the nucleus and enter the cytoplasm. What's next for the RNA molecule? More on transcription.
How are polymerases different in prokaryotes and eukaryotes? How is bacterial transcription unique? How is transcription regulated? Once an mRNA molecule is complete, that molecule can go on to play a key role in the process known as translation. During translation , the information that is contained within the mRNA is used to direct the creation of a protein molecule.
In order for this to occur, however, the mRNA itself must be read by a special, protein-synthesizing structure within the cell known as a ribosome. Watch this video for a summary of eukaryotic transcription. What are introns and exons?
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