What is the dideoxy method?
In the basic dideoxy sequencing reaction, an oligonucleotide primer is annealed to a single-stranded DNA template and extended by DNA polymerase in the presence of four deoxyribonucleoside triphosphates (dNTPs), one of which is 35S-labeled.
How do you read a dideoxy sequencing gel?
The bands of the gel are detected, and then the sequence is read from the bottom of the gel to the top, including bands in all four lanes. For instance, if the lowest band across all four lanes appears in the A reaction lane, then the first nucleotide in the sequence is A.
How does dideoxy sequencing work?
Sanger sequencing results in the formation of extension products of various lengths terminated with dideoxynucleotides at the 3′ end. The extension products are then separated by Capillary Electrophoresis or CE. The molecules are injected by an electrical current into a long glass capillary filled with a gel polymer.
Why is it called the dideoxy method?
The dideoxy method gets its name from the critical role played by synthetic nucleotides that lack the -OH at the 3′ carbon atom (red arrow). Because AZT has no 3′ -OH group, DNA synthesis by reverse transcriptase halts when AZT triphosphate is incorporated in the growing DNA strand.
How does gel electrophoresis analyze DNA?
Gel electrophoresis is a technique used to separate DNA fragments according to their size. DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel. DNA fragments are negatively charged, so they move towards the positive electrode.
What is the function of DNA sequencers?
DNA sequencing is a laboratory technique used to determine the exact sequence of bases (A, C, G, and T) in a DNA molecule. The DNA base sequence carries the information a cell needs to assemble protein and RNA molecules. DNA sequence information is important to scientists investigating the functions of genes.
What is the purpose of DdNTPs in DNA sequencing?
The Sanger method is used to amplify a target segment of DNA, so that the DNA sequence can be determined precisely. The incorporation of ddNTPs in the reaction valves are simply used to terminate the synthesis of a growing DNA strand, resulting in partially replicated DNA fragments.
How is dideoxy used in in vitro DNA synthesis?
The key element of the method is the use of dideoxyribonucleoside triphosphates (ddNTPs) 1 in in vitro DNA synthesis mixtures. Figure 1 compares the structure of a regular dNTP and that of a ddNTP.
How is capillary gel electrophoresis used in DNA sequencing?
Capillary gel electrophoresis has been demonstrated for the separation and detection of DNA sequencing samples. Enzymatic dideoxy nucleotide chain termination was employed, using fluorescently tagged oligonucleotide primers and laser based on-column detection (limit of detection is 6,000 molecules p …
How is primer extended in presence of dideoxynucleotide?
Primer will be extended in the presence of dideoxynucleotides that will promote termination of the extension reaction (see Sanger Dideoxy Sequencing of DNA). Reactions will be treated with base to hydrolyze any RNA that may remain following reverse transcription and neutralized prior to electrophoresis.
How to do a problem solving test with dideoxy sequencing?
After electrophoresis the gel was dried and exposed to an X-ray film. Figure 2 shows the autoradiogram of the gel. Study the description of the experiment and the results and solve the following multiple-choice questions (MCQs). (The following statements are related to the information presented in the description of the experiment.