Which of the following statements is true regarding the use of oligonucleotide primers in PCR?
A. Oligonucleotide primers must hybridize completely to the template DNA strand in order for PCR to proceed; nonhybridized DNA will be degraded.
B. The use of oligonucleotide primers is obsolete with the Gibson method of seamless cloning; instead, DNA polymerase and DNA ligase are able to copy the DNA independently.
C. Oligonucleotide primers must hybridize to the 5' end of the template strand leaving a 3' primer "overhang"; that sequence will be copied and incorporated into the daughter strands allowing seamless cloning to occur.
D. Only a portion of the oligonucleotide primers need to hybridize to the 3' template DNA strand; the 5' "overhang" can contain a restriction site that will be copied during the second and all subsequent cycles of PCR.
Answer: D
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