Extraction Reagents and Complimentary Deoxyribonucleic Acid Preparation

1. Ficoll-Hypaque (Amersham Pharmacia; Buckinghamshire, UK).

2. TRI reagent.

4. Ethanol.

5. Isopropanol, all molecular biology grade, (Sigma-Aldrich; Poole, Dorset, UK).

6. RPMI 1640 medium/bovine fetal calf serum (Gibco Invitrogen, Paisley, UK).

7. Dimethylsulfoxide (Sigma).

8. Wizard Genomic DNA Purification Kit (Promega, Southampton).

9. First-strand cDNA synthesis kit (Amersham Pharmacia).

Fig. 1. A schematic representation of an immunoglobulin (Ig) molecule. Both the Ig variable (V) and constant (C) regions are shown for the heavy (VH / CH) and light (VL / CL) chains. The VH region or domain has been further delineated to illustrate the contribution of germline VH, DH, and JH gene segments in assembly of the functional V(D)J unit. Note that the mutational status is determined solely from the VH gene segment as indicated.

Fig. 1. A schematic representation of an immunoglobulin (Ig) molecule. Both the Ig variable (V) and constant (C) regions are shown for the heavy (VH / CH) and light (VL / CL) chains. The VH region or domain has been further delineated to illustrate the contribution of germline VH, DH, and JH gene segments in assembly of the functional V(D)J unit. Note that the mutational status is determined solely from the VH gene segment as indicated.

CLL: Unmutated VH ctene

QVQLVQSGAEVKKPGASVKVSCKASGYTFT VI-02 CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG GCC TCA GTG AAG GTC TCC TGC AAG GCT TCT GGA TAC ACC TTC ACC

|-------CDRl-------| |-------------------------------CDR2--------

GYYMHWVRQAPGQGLEWMGWINPNSGGTNY VI-02 GGC TAC TAT ATG CAC TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT GAG TGG ATG GGA TGG ATC AAC CCT AAC AGT GGT GGC ACA AAC TAT

AQKFQGWVTMTRDTS I STAYMELSRLRSDD VI-02 GCA CAG AAG TTT CAG GGC TGG GTC ACC ATG ACC AGG GAC ACG TCC ATC AGC ACA GCC TAC ATG GAG CTG AGC AGG CTG AGA TCT GAC GAC

TAVYYCAR VI-02 ACG GCC GTG TAT TAC TGT GCG AGA

CLL: mutated Vh gene

EVQLVESGGGLVQPGGSLRLSCAASGFTFS V3-7 GAG GTG CAG CTG GTG GAG TCT GGG GGA GGC TTG GTC CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTT AGT

|-------CDRl-------| |--------------------------------CDR2-------

SYWMSWVRQAPGKGLEWVANIKQDGSEKYY V3-7 AGC TAT TGG ATG AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTG GCC AAC ATA AAG CAA GAT GGA AGT GAG AAA TAC TAT

VDSVKGRFTI SRDNAKNSLYLQMNSLRAED V3-7 GTG GAC TCT GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AAC GCC AAG AAC TCA CTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAG GAC

TAVYYCAR V3-7 ACG GCT GTG TAT TAC TGT GCG AGA

Fig. 2. Tumor-derived VH genes from an unmutated and mutated CLL case. Each sequence is shown aligned at the nucleotide level with the germline VH gene segment to determine mutational status. For the mutated CLL case shown, there is a 89.1% homology to the germline V3-7 gene, identified as donor by a best-fit homology match from the databases.

2.2. Primers

1. MWG Biotech; Milton Keynes (www.mwg-biotech.com).

2.3. PCR Reagents

1. Hotstart Taq DNA polymerase (QIAGEN; Crawley, West Sussex, UK).

3. Agarose (Gibco Invitrogen).

4. Ethidium bromide (Gibco Invitrogen).

5. Hybaid recovery DNA purification kit II (Hybaid; Ashford, Middlesex, UK).

2.4. Cloning Reagents

1. pGEM T vector kit (Promega; Southampton, UK).

2. E. coli strain JM109 competent cells (Promega).

3. Ampicillin (SmithKline Beecham Pharmaceuticals Ltd; Laoghaire, Co. Dublin).

4. LB (Millers Luria broth), agar (used at 1.5% w/v; Sigma-Aldrich).

5. X-Gal, 50 mg/mL (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside-Promega; Madison, WI).

6. Isopropyl-ß-D-thio-galactopyranoside (IPTG; Calbiochem; Beeston, Nottingham, UK).

7. Spin Miniprep kit (QIAGEN).

2.5. DNA Sequence Analysis

1. Big dye version 1 (Applied Biosystems; Warrington, Cheshire, UK).

2. ABI 377 DNA sequencer (Applied Biosystems).

3. Sodium acetate, molecular biology grade (Sigma).

2.6. Software

1. MacVector (Oxford Molecular Group plc).

2. V base (MRC Centre for Protein Engineering; Cambridge, UK).

3. Methods

3.1. Patient Samples

1. Obtain 10-20 mL of peripheral blood (PB) by venepuncture and put into precoated heparinized or ethylenediamine tetraacetic acid tubes, for processing on the same day.

2. PB mononuclear cells (PBMCs) are obtained by standard Ficoll-hypaque density centrifugation.

3. PBMCs can be stored as pellets (approx 5 x 106) at -80°C or in dimethylsulfox-ide (DMSO) containing storage medium (RPMI + 50% FCS + 10% DMSO) for ribonucleic acid (RNA).

4. For samples stored in DMSO, a wash step using sterile medium will be required before the extraction of RNA.

5. For DNA, cell pellets can be stored at 20°C. An aliquot of PB can be stored directly at -20°C for subsequent isolation of DNA. DNA may be isolated from transit samples within 48 h.

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