Multiplex PCR Amplification of Tcrb Tcrg Tcrd and Control Genes

3.3.1. PCR Amplification of TCRG Chain Genes

1. Two multiplex PCR tubes are used to analyze different VJ recombinations of the TCRG chain genes. Both tube A and tube B are set up similarly with the same J primers but different V primers (Fig. 2). The J1.3/2.3 primer is labeled with FAM, and the J1.1/2.1 is labeled with HEX (see Note 7). The tubes (see Note 8) are set up (in a total reaction volume of 50 |L) as follows:

TCRG mix

Sterile water (see Note 9) 10X buffer II dNTP mix (1.25 mM) MgCl2 (25 mM) TCRG primers" AmpliTaq Gold 100 ng of DNA

aTCRG tube A: VF1, V10, J1.3/2.3, J1.1/2.1; for "TCRG tube B: V9, V11, J1.3/2.3, J1.1/2.1. For storage of PCR reagents, see Notes 10 and 11, for general PCR set up, and for validity of data, see Notes 12-20.

Pcr Analysis

Fig. 2. PCR analysis of TCRG gene rearrangements. (A) Schematic diagram indicating the appropriate position and designation of V and J region primers for TCRG PCR analysis. The hatched boxes represent N regions. (B) Heteroduplex analysis of PCR products from both tube A and B PCRs. M, molecular weight marker; P, polyclonal peripheral blood DNA; C, clonal T-cell lymphoma DNA. Such analysis exploits the differences in the sizes of the PCR products as well as the variation in sequences. Heteroduplexes of imperfectly matched junctional sequences are seen as ill defined smears retarded in the gel whereas homoduplexes of identical junc-tional sequences are identified as narrow bands within the appropriate size range. (C) Genescan analysis of the same samples as shown in (B). PCR fragment size is plotted against fluorescent intensity. TCRG analysis of polyclonal samples for both tube A and B demonstrate four Gaussian distributions representing different gene recombinations in varying magnitudes. For tube A, PCR analysis identifies a clonal population (narrow peak) representing Vf1-J1.3/2.3 gene recombination. A different clonal sample for tube B analysis demonstrates a V9-J1.3/2.3 gene recombination. See Color Plate 6, following page 270.

2. The standardized cycling conditions are: 94°C for 10 min, then 35 cycles of 94°C for 1 min/60°C for 1 min/72°C for 1 min. The samples are then heated to 72°C for 10 min for a final extension, prior to holding at 8°C.

3.3.2. PCR Amplification of TCRB Chain Genes

1. Three multiplex PCR tubes are required to analyze partial and complete TCRB chain gene rearrangements (Fig. 3). All JB primers are labeled with FAM or for distinction between different J gene usage JB1 primers can be labeled with FAM and JB2 primers with HEX (see Note 7). The PCRs are set up (in a total reaction volume of 50 ^L) as follows (see Note 8):

TCRB-A mix TCRB-B mix

Sterile water (see Note 9)

27.4 ^L

27.9 ^L

10X buffer II

5 pL

5 pL

dNTP mix (1.25 mM)

8 ^L (0.2 mM)

8 ^L (0.2 mM)

MgCl2 (25 mM)

6 ^L (3.0 mM)

6 ^L (3.0 mM)

TCRB primers

3.2 ^L (10 pmol

2.7 ^L (10 pmol

of each primer3)

of each primer6)

AmpliTaq Gold

0.4 ^L (2U)

0.4 ^L (2 U)

100 ng DNA

TCRB-C mix

Sterile water (see Note 9) 33.2 (L

10X buffer II 5 (L

TCRB primers 1.5 (L (10 pmol of each primerc)

100 ng DNA

<TCRB A mix (32 primers): 23 VB primers, 6 JB1 primers (JB1.1-1.6), and 3 JB2 primers (JB2.2, 2.6, 2.7).

6TCRB B mix (27 primers): 23 VB primers and 4JB2 primers (JB2.1, 2.3, 2.4, 2.5).

CTCRB C mix (15 primers): DB1, DB2 primers and 6 JB1 primers (JB1.1-1.6)

and 7 JB2 primers (JB2.1-2.7). For storage of PCR reagents, see Notes 10 and 11, and for general PCR set up and validity of data, see Notes 12-20.

2. The PCR cycling conditions are as noted previously. 3.3.3. PCR Amplification of TCRD Chain Genes

1. Only one multiplex PCR tube is necessary to analyze full and partial TCRD gene rearrangements (including VDJ, DJ, DD, and VD). There are six V primers (VD1, VD2, VD3, VD4, VD5, VD6), three J primers (JD1, JD2, JD3), and two D prim-

Multiplex Pcr Analysis Str

Fig. 3. PCR analysis of TCRB gene rearrangements. (A) Schematic diagram indicating the appropriate position and designation of V, D, and J region primers for the analysis of complete and partial TCRB gene rearrangements. The hatched boxes represent N regions. (B) Heteroduplex analysis of PCR products from tube A and C PCR reactions. M, molecular weight marker; P, polyclonal peripheral blood DNA; C, clonal T-cell lymphoma DNA. (C) Genescan analysis of the same samples as shown in B. TCRB analysis of polyclonal peripheral blood DNA identifies one Gaussian distribution in tube A and two in tube C. A complete TCRB VDJ rearrangement is identified in the clonal sample for tube A analysis whereas a clonal peak identifying a D2-J recombination is identified for tube C analysis. See Color Plate 7, following page 270.

Fig. 3. PCR analysis of TCRB gene rearrangements. (A) Schematic diagram indicating the appropriate position and designation of V, D, and J region primers for the analysis of complete and partial TCRB gene rearrangements. The hatched boxes represent N regions. (B) Heteroduplex analysis of PCR products from tube A and C PCR reactions. M, molecular weight marker; P, polyclonal peripheral blood DNA; C, clonal T-cell lymphoma DNA. (C) Genescan analysis of the same samples as shown in B. TCRB analysis of polyclonal peripheral blood DNA identifies one Gaussian distribution in tube A and two in tube C. A complete TCRB VDJ rearrangement is identified in the clonal sample for tube A analysis whereas a clonal peak identifying a D2-J recombination is identified for tube C analysis. See Color Plate 7, following page 270.

Tcrg Rearrangement

Fig. 4. PCR analysis of TCRD gene rearrangements. (A) Schematic diagram indicating the appropriate position and designation of V, D, and J region primers for complete (VDJ) and partial (DJ, DD, VD) TCRD PCR analysis. The hatched boxes represent N regions. (B) Heteroduplex analysis of PCR products from the single multiplex tube used for TCRD analysis. M, molecular weight marker; P, polyclonal peripheral blood DNA; C, clonal T-cell lymphoma DNA. (C) Genescan analysis of the same samples as shown in B. The fluorescent profile of a polyclonal sample is distinct from a normal Gaussian distribution (seen in Figs. 2C and 3C) as a consequence of the large range in TCRD PCR product sizes. However, clonal products are easily identified as shown for the representative clonal DNA from a T-cell lymphoma showing a partial DJ and full VDJ clonal rearrangement. See Color Plate 8, following page 270.

Fig. 4. PCR analysis of TCRD gene rearrangements. (A) Schematic diagram indicating the appropriate position and designation of V, D, and J region primers for complete (VDJ) and partial (DJ, DD, VD) TCRD PCR analysis. The hatched boxes represent N regions. (B) Heteroduplex analysis of PCR products from the single multiplex tube used for TCRD analysis. M, molecular weight marker; P, polyclonal peripheral blood DNA; C, clonal T-cell lymphoma DNA. (C) Genescan analysis of the same samples as shown in B. The fluorescent profile of a polyclonal sample is distinct from a normal Gaussian distribution (seen in Figs. 2C and 3C) as a consequence of the large range in TCRD PCR product sizes. However, clonal products are easily identified as shown for the representative clonal DNA from a T-cell lymphoma showing a partial DJ and full VDJ clonal rearrangement. See Color Plate 8, following page 270.

ers (one 5' D2 and one 3' D3; Fig. 4; see Note 7). To identify different partial and full TCRD recombinations, JD1 is labeled with HEX, JD2-JD4 with FAM, and 3'D3 with NED. The PCR set up (in a total reaction volume of 50ul) as follows (see Note 8):

TCRD mix

Sterile water (see Note 9) 10X buffer II dNTP mix (1.25 mM) MgCl2 (25 mM) TCRD primer mix AmpliTaq Gold 100 ng DNA

For storage of PCR reagents, see Notes 10 and 11, and for general PCR set up and validity of data, see Notes 12-20.

2. The standardized cycling conditions as noted previously are used.

3.3.4. PCR Amplification of Control Genes

To assess DNA quality and amplifiability, PCR is performed on the specimen DNA with primer pairs designed to amplify amplicons of 100, 200, 300, and 400bp (and 600 bp if required).

1. One multiplex PCR is required to analyze the control genes. Primer mix A contains 0.25 ||L of forward and reverse primers for the following genes: TBXAS1 (exon 9), RAG1 (exon 2), PLZF (exon 1), and AF4 (exon 11) to give amplicons of 100, 200, 300, and 400 bp, respectively. This PCR is suitable for assessment of DNA extracted from paraffin-embedded samples. Primers that amplify a fragment of 600 bp can be added if required for high-quality DNA (mix B). The PCR reaction is set up as follows (see Note 8):

Control tube mix A

Sterile water (see Note 9) 29.8 |L

10X Buffer II 5 |L

Primer mix A 2.0 |L (2.5pmol of each primer)

100 ng DNA

If required, 0.5 mL (2.5 pmol) of forward and reverse primers for AF4 (exon 3) can be added to amplify amplicons of 600 bp to the above reaction mix and is then designated mix B.

For storage of PCR reagents, see Notes 10 and 11, and for general PCR set up, see Notes 12-19.

2. The standardized cycling conditions are used as noted previously.

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