Science · 1989 · 632 citations · 25 references
Life SciencesImmunologyMolecular BiologyCellular ImmunologyImmunogeneticsMolecular ImmunologyCell-type Specific Gene ExpressionPolymerase Chain ReactionT Cell BiologyT Cell DifferentiationT Cell Receptor SignalingT-cell Receptor GeneticsT-cell Receptor Delta Chain GeneticsT-cell Receptor Gene ExpressionT-cell Receptor Gene SegmentsT-cell Receptor Gene Usage
PCR uses two primers to amplify sequences between them, but this approach fails when the 5′ sequence of the target is unknown, as in T cell receptor or immunoglobulin genes. The authors aimed to develop anchored PCR, a method that requires sequence specificity only at the 3′ end of the target fragment. Anchored PCR was employed to analyze TCR δ chain mRNA from human peripheral blood γδ T cells. The analysis revealed a previously undescribed Vδ3 gene segment in most cells and a distinct δ‑chain junctional sequence subpopulation relative to earlier reports.
In the polymerase chain reaction (PCR), two specific oligonucleotide primers are used to amplify the sequences between them. However, this technique is not suitable for amplifying genes that encode molecules where the 5′ portion of the sequences of interest is not known, such as the T cell receptor (TCR) or immunoglobulins. Because of this limitation, a novel technique, anchored polymerase chain reaction (A-PCR), was devised that requires sequence specificity only on the 3′ end of the target fragment. It was used to analyze TCR δ chain mRNA's from human peripheral blood γδ T cells. Most of these cells had a Vδ gene segment not previously described (V δ 3), and the δ chain junctional sequences formed a discrete subpopulation compared with those previously reported.
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Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase
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