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MOLECULAR BIOLOGY: WORKING WITH DNA
POLYMERASE CHAIN REACTION
IDENTIFYING DIFFERENT CLONES OF scFv USING POLYMERASE CHAIN REACTION
Identifying Different Clones of scFv Using Polymerase Chain Reaction
Contributor: The Laboratories of Andrew Bradbury at Los Alamos National Laboratory and James D. Marks University of California, San Francisco Overview
When an ELISA analysis (see Protocol ID# 2211 and Protocol ID#2209) indicates that a number of antigen-binding antibodies have been selected, PCR can be used to estimate the number of different clones present. In this protocol, the scFv gene is amplified directly from bacterial colonies harboring the phagemid by amplifying with primers that flank the scFv gene. The PCR products are digested with a restriction enzyme that cuts frequently within V-genes (e.g., BstN1). Procedure
A. PCR Amplification of scFv
1. Prepare a PCR Master Mix solution (volumes are per reaction, also see Hint #1):
15.0 μl of ddH2O
2.0 μl of 10X Taq Polymerase Buffer
1.6 μl of 25 mM MgCl2
1 μl of 20X dNTP Mix
1 μl of 5' DNA Primer (10 μM)
1 μl of 3' DNA Primer (10 μM)
0.05 μl of Taq DNA Polymerase
2. Aliquot 20 μl of the PCR Master Mix into 0.5 ml microcentrifuge tubes or into 96 well PCR microplates.
3. Using a sterile toothpick, gently touch a single colony and swirl the colony into a single PCR reaction mixture. Discard the toothpick or, if required, use the excess colonies to rescue the colony into medium or onto an agar plate by touching the surface (see Hint #2).
4. Overlay each PCR Reaction with Mineral Oil (Sigma).
5. Incubate the PCR Reaction for 10 min at 94°C with a PCR-Block (see Hint #3).
6. Amplify the Selected Genes in a thermal cycling block as follows:
Cycle 30 Times
94°C for 1 min
55 to 60 °C for 1 min
72 °C for 1 min (2 min for Fabs)
7. The contributor of the protocol suggests checking 5 μl of the PCR Reaction on a 2% TBE Agarose Gel (see Protocol ID#1265). The analysis will indicate how many clones contain full-length inserts.
8. Prepare the following Restriction Mix (volumes are per reaction):
0.2 μl of Acetylated BSA
4 μl of 10X BstNI Buffer (NEB)
15.5 μl of ddH2O
0.5 μl of BstNI (10 Units/μl, NEB)
9. Add 20 μl of Restriction Mix to each of the PCR Reactions (see Hint #4).
10. Incubate the mixture at 60°C for 2 to 3 hr.
11. Combine 2 μl of Sample Dye with 8 μl of restriction digested sample (from under the mineral oil) and mix well by flicking.
12. Electrophoreses the sample through a 4% Nusieve Agarose Gel cast in 0.5 X TBE containing 0.05 μl/ml Ethidium Bromide (see Hint #5).
13. Electrophorese the sample at 100 V (10 V/cm) and compare the band patterns of individual clones on a UV transilluminator (see Hint #6).
These primers are described for PCR fingerprinting and DNA sequencing. (see Hint #7)
5'-CAG GAA ACA GCT ATG AC-3'
5'-GAA TTT TCT GTA TGA GG-3'
5'-CGA TCC GCC ACC GCC AGA-3'
5'-CTA TGC GGC CCC ATT CA-3'
3' DNA Primer 10 μM Oligonucleotide (see Section B) 5' DNA Primer 10 μM Oligonucleotide (see Section B) Acetylated BSA 10 mg/ml Acetylated Bovine Serum Albumin 25 mM Magensium Chloride TBE Buffer (10X) 890 mM Boric Acid
890 mM Tris
20 mM EDTA, pH 8.0
dNTP Mix (20X) 5 mM dCTP
5 mM dGTP
5 mM dATP
5 mM dTTP
Taq Polymerase Buffer (10X) 15 mM MgCl2
500 mM KCl
Any brand of Taq polymerase in its appropriate buffer can be used
100 mM Tris-HCl, pH 9.0 at 25°C
1 % Triton X-100
BioReagents and Chemicals
DNA Restriction Enzyme, BstNI
DNA Polymerase, Taq
Bovine Serum Albumin, Acetylated
1. If the PCR buffer does not contain Mg, it should be added to a final concentration of 1.5 mM. Alternatively, use the PCR reagents provided with the DNA polymerase.
2. Excess bacteria in the PCR reaction can inhibit the PCR reaction. Additionally, 1 to 2 μl of a Glycerol Stock or the Master Plate Culture can be used instead of a colony.
3. The initial 94°C incubation is required to break open the bacteria and release the template DNA.
4. Make sure to add the Restriction Mix to the solution that is beneath the Mineral Oil.
5. Protocol ID#1265 can be adapted using Nusieve Agarose in place of Agarose. Alternatively, the sample can be electrophoresed through a 2.5% Agarose gel; however, the resolution will not be as good as what can be achieved with Nusieve Agarose.
6. scFv with different restriction patterns represent unique clones. However, scFv with the same pattern may also represent different clones (approximately 10% of the time). This results from the fact that BstN1 (and other enzymes) generally cut V-genes in the framework regions. This assay may not be as informative on libraries with more restricted diversity (e.g. immune libraries, affinity maturation libraries, etc.) since the diversity in these libraries may not translate into gain or loss of restriction sites.
7. Please check the sequences of these primers against the sequence of pHEN 1 to determine which primer combinations to use to amplify the scFv genes. Also see Complete Primer Sequences.