Screening for Full Length Inserts Using PCR and for VH Gene Diversity Using DNA Fingerprinting

In this procedure, the positive clones will be analyzed for the presence of DNA (about 1 kbp) encoding the scFv antibody. In addition, as an initial screening for the diversity of the clones, restriction enzyme analysis will be performed. (Also see Fig. 2).

Fig. 2. Restriction fragment analysis of clones expressing anti-heparan sulfate antibodies. Polymerase chain reaction (PCR) is preformed to amplify the region encoding the scFv antibody (A), using a set of primers flanking the VH and VL segments. The resulting (full-length) PCR fragments (B) harbor a unique pattern of BstNl cleavage sites (CC*A/TGG), serving as a fingerprint. Following digestion with restriction enzyme BstNl (C), the fragments are separated on an agarose gel and the restriction patterns of each clone are compared (D). Clones with unique restriction patterns are selected and sequenced to establish the VH family, germ line segment (DP number, see ref. 4) and VH -CDR3 sequence (randomized in the library used; see ref. 3).FR, framework region; CDR, complementary determining regions; M, DNA marker.

Fig. 2. Restriction fragment analysis of clones expressing anti-heparan sulfate antibodies. Polymerase chain reaction (PCR) is preformed to amplify the region encoding the scFv antibody (A), using a set of primers flanking the VH and VL segments. The resulting (full-length) PCR fragments (B) harbor a unique pattern of BstNl cleavage sites (CC*A/TGG), serving as a fingerprint. Following digestion with restriction enzyme BstNl (C), the fragments are separated on an agarose gel and the restriction patterns of each clone are compared (D). Clones with unique restriction patterns are selected and sequenced to establish the VH family, germ line segment (DP number, see ref. 4) and VH -CDR3 sequence (randomized in the library used; see ref. 3).FR, framework region; CDR, complementary determining regions; M, DNA marker.

1. Select heparan sulfate-positive clones (as detected by ELISA) from the corresponding master plate (see Subheading 3.2.). Plate bacteria on 94/15 dishes with TYE containing 100 ^g of ampicillin/mL and 1% (w/v) glucose to obtain single colonies.

2. Pick a single colony of each clone (mark the colony on the back of the Petri dish) with a sterile toothpick and transfer the cells into the following PCR-mixture: 34.5 ^L H2O, 5.0 ^L 10X PCR buffer, 2.5 ^L 20X dNTP (5 mM each), 2.5 ^L LMB3 primer (10 pmol/^L), 2.5 ^L fd-SEQ1 primer (10 pmol/^L), 0.5 ^L Tag polymerase (5 U/^L). Overlay the PCR mixture with a droplet of mineral oil and use the following PCR program: 10 min at 94°C, 30 cycles of 1 min at 94°C, 1 min at 60°C, 2 min at 72°C, 10 min at 72°C, cool to 4°C.

3. Take 4 ^L of the PCR-mixture and add 1 ^L of 5X DNA sample buffer. Run the samples on a 1% (w/v) Seakem agarose gel (add 3.0 ^L ethidium bromide [10 mg/mL] to 75 mL of agarose solution). Include DNA marker (250 ng) in one of the lanes. Run the gel at 50 V in an appropriate volume of 1X TBE buffer. Analyze the gel on a UV transilluminator. PCR products of about 1000 base pairs indicate full-length clones (see Note 15).

4. For DNA fingerprinting take 20 ^L of the PCR-mixture and add 20 ^L of the following restriction enzyme mix: 17.8 ^L H2O, 2.0 ^L 10X NEbuffer 2, 0.2 ^L BstNI (10 U/^L). Overlay with mineral oil and incubate at 60°C for 3 h. Take 8 ^L and add 2 ^L 5X DNA sample buffer. Run the restriction mixtures on a 4% (w/v) 3/1 NuSieve agarose gel as described in step 3, Subheading 3.4., Differences in banding pattern indicate different clones (see Note 16 and Fig. 2).

5. Take with a sterile toothpick unique clones from the plate with marked colonies (step 2, Subheading 3.4.) and add to 10 mL 2XTY containing 100 ^g ampicillin/mL and 1% (w/v) glucose. Grow for 16-20 h, while shaking, at 37°C.

6. Take 1.5 mL of the bacterial culture (step 5, Subheading 3.4.) for preparation of phagemid DNA, used for sequencing and for long-term storage (see Note 17). For phagemid isolation and sequencing we use materials described in steps 13 and 14, Subheading 2.4.

7. Spin the rest of the culture at 3000g for 10 min at 4°C. Decant the supernatant and resuspend the pellet in 1 mL of ice-cold 2XTY containing 15% (v/v) glycerol. Aliquot the bacterial suspension into several sterile cryovials and store at -70°C.

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