Dideoxy method

FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

Figure 28.8 The sequence of a restriction fragment determined by the Sanger dideoxy method can be read simply by noting the colors of the dye attached to each of the various terminal nucleotides.

So efficient is the automated dideoxy method that sequences up to 1100 nucleotides in length, with a throughput of up to 19,000 bases per hour, can be sequenced with 98% accuracy. After a decade of work, preliminary sequence information for the entire human genome of 2.9 billion base pairs was announced early in 2001- Remarkably, our genome appears to contain only about 30,000 genes, less than one-third the previously predicted number and only twice the number found in the common roundworm. 

Sequencing of DNA is carried out by the Sanger dideoxy method, and small DNA segments can be synthesized in the laboratory by automated instruments. Small amounts of DNA can be amplified by factors of 106 using the polymerase chain reaction (PCR). 

Sanger dideoxy method (Section 2.6) The most commonly used method of DNA sequencing. 

The Klenow fragment. The DNA polymerase 1 molecule contains its polymerase and nuclease activities on different parts of the enzyme molecule. These two parts can be separated by treatment with the enzyme sub-tilisin. The part which retains the polymerase function is known as the Klenow fragment. This enzyme sythesizes a new DNA strand complementary to the single strand of DNA (the template) only. It is used to create blunt ends in dsDNA and in the dideoxy method of DNA sequencing. 

Which of the following statements are true of the dideoxy method for... 

Correct answer = B. The sequence of the new strand of DNA synthesized using the Sanger dideoxy method may be determined by reading the bands from the bottom to the top of the gel as 5TACCAG3 This is complementary to the original strand, which is. therefore, 5 CTGGTA3 ... 

Common method of DNA sequencing Cloned, purified fragments of DNA can be sequenced using the Sanger dideoxy method. 

The primer extension assay is similar to the dideoxy method commonly used in sequence analysis. In the primer extension assay, a region containing the mutation to be assayed is amplified in a first PCR reaction. A specific primer, which is designed to anneal directly upstream of the base which is the site of a known mutation, is then used in a second reaction. Radioac-tively labeled dideoxy nucleotides corresponding to either the normal or the mutant base at the potential mutation site are added in separate tubes. During the reaction, the labeled nucleotide added to the 3 end of the primer will depend on the sequence at the potential mutation site. If the normal sequence is present at the potential mutation site, only the reaction containing the normal labeled dideoxy nucleotide will produce a labeled primer. Conversely, if the mutant sequence is present at the mutation site, only... 

Currently the dideoxy method probably is the method of choice for sequencing DNA by primed synthesis methods and is generally applicable to any DNA that can be obtained in single-stranded form. In the last two years two important advances have been made which have essentially solved the problem of preparing the DNA template in single-stranded form and these are described in the following Section and in Chapter 4. The first method, which... 

What are the requirements for sequencing DNA with the Sanger dideoxy method ... 

The most common method is called chain-termination sequencing or the dideoxy method, and is described in Chap. 16. 

A number of nucleotide analog compounds function by blocking further chain growth at the replication fork. The 2 3 -dideoxynucleosides can be converted to the triphosphates (ddNTPs). In the case of bacteria, these are incorporated onto the 3 -hydroxyl end of a growing DNA chain, and because the new end now lacks a 3 hydroxyl, no further additions can occur. They are used in conjunction with DNA polymerase I in the dideoxy method of Sanger for determining DNA sequences. 

The dideoxy method of Sanger for sequencing DNA involves the copying of a single strand of the DNA by a DNA polymerase (DNA polymerase I was originally used) to yield new strands (radioactively labeled for identification purposes), which are terminated at certain positions through the incorporation of a dideoxy nucleotide at the 3 end. A short primer, complementary to a sequence at one end of the single-strand template. 

DNA Sequence Analysis. DNA from AgtlO clone 14-1 was digested with restriction endonuclease EcoRI and cloned into M13 mpll (18). The bacteriophage M13 derivatives were constructed using methods described by Messing (19). Both orientations of the inserts were represented in independent clones that were sequenced by the dideoxy method (20). 

DNA sequencing Maxam-Gilbert method restriction endonuclease restriction fragment palindrome Sanger dideoxy method DNA synthesis DMT ether phosphoramidite phosphite polymerase chain reaction (PCR)... 

Sanger dideoxy method (Section 28.6) an enzymatic method for DNA sequencing. 

Figure 5-21. DNA sequencing by the Sanger-dideoxy method. Single or double stranded DNA is used as a template for a primer-dependent DNA polymerase extension, which is carried out in four separate reaction mixes. In addition to DNA polymerase and the four dNTPs (including an a-radiolabelled nucleoside triphosphate), each reaction mix contains a small proportion of one dideoxy nucleotide derivative, ddATP,...

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