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The determination of the order of the four bases—adenine, thymine, cytosine, and guanine—in a strand of DNA is termed __________.
DNA sequencing is used to determine the exact order of nucleotides that compose a given DNA molecule. This process can refer to any method that is used to determine the order of the nitrogenous bases in a strand of DNA.
DNA amplification is the duplication of regions of DNA to form multiple copies of a specific portion of the original region. DNA replication is the process of producing two identical replicas from one original DNA molecule. Gene flow is the movement of alleles from one population to another, owing to migration of individual organisms. Genetic recombination is the generation of new combinations of alleles on homologous chromosomes due to the exchange of DNA during crossing over.
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Which of the following accounts for the inability of dideoxynucleotide triphosphates to further polymerize in Sanger DNA sequencing?
Dideoxynucleotide triphosphates (ddNTPs) lack a 3' hydroxyl group, which is necessary for further polymerization past the ddNTP. In the absence of this 3' free hydroxyl group, ddNTPs cannot be linked to the next incoming nucleotide's 5' end. This halts DNA synthesis, and is the basis of Sanger (chain-termination) sequencing. Ordinary nucleotides do possess a 3' hydroxyl group, allowing them to polymerize further. Sanger sequencing takes advantage of this difference to terminate synthesis at various points.
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Traditional Sanger style DNA sequencing relies on a method called chain termination. What type of molecule is used to terminate DNA chains to create molecules of all possible lengths covering the fragment to be sequenced?
The key to the chain termination method used in Sanger sequencing is the dideoxynucleotide. These modified bases, designated as ddNTP's rather than dNTP's, are missing their 3' hydroxyl group. This prevents further elongation of DNA strands being synthesized by a polymerase since DNA polymerases all require a 3' hydroxyl as their substrate. In the context of a whole sequencing reaction done before actual DNA sequencing, this creates strands of all possible lengths along your desired sequence.
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