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MOLECULAR BIOLOGY: WORKING WITH DNA
POLYMERASE CHAIN REACTION
PCR GENE SPLICING BY AMPLIFICATION OF VH AND VL GENES TO CREATE scFv GENE REPERTOIRES
PCR Gene Splicing by Amplification of VH and VL Genes to create scFv Gene Repertoires
Contributor: The Laboratories of Andrew Bradbury at Los Alamos National Laboratory and James D. Marks University of California, San Francisco Overview
This protocol describes the creation of an scFv library of DNA fragments with PCR. In this procedure, pools of fragments corresponding to the heavy and light chains of antibody variable (V) regions, as well as a "linker" fragment consisting of repeated glycine/serine encoding DNA, are randomly joined together. After several rounds of amplification, the subsequent spliced products are amplified with primers flanking the 5' and 3' ends of the joined fragments. PCR splicing can be accomplished with three (VH and VL and linker region) or two (VH and VL) DNA fragments (see Hint #6). The latter is not feasible when assembling Fab fragments because the size of the linker is too large. Furthermore, in two-fragment assembly for scFv, the repetitive nature of the linker needs to be overcome by physically extending the oligonucleotides on one side so that they extend into the other V-gene or by amplifying from cloned material (see Image #1). The protocol below is for 3-fragment assembly. Procedure
A. Polymerase Chain Reaction
1. Prepare the following PCR reaction mixtures in 0.5 ml microcentrifuge tubes (see Hint #1):
1.0 μl of 100 ng/μl ScFv Linker DNA (prepared in Protocol ID#2197)
3.5 μl of 100 ng/μl of VH Repertoire DNA
3.0 μl of 100 ng/μl of VL Repertoire DNA (Vκ or Vλ)
Control Tube #1
1.0 μl of 100 ng/μl ScFv linker DNA
6.5 μl of ddH2O
Control Tube #2
1.0 μl of 100 ng/μl of VH Repertoire DNA
1.0 μl of 100 ng/μl of VL Repertoire DNA (Vκ or Vλ)
4.5 μl of ddH2O
2. Add the following components to each tube:
30.5 μl of ddH2O
5.0 μl of 10X Vent Polymerase Buffer
5.0 μl of 20X dNTP Mix
3. In a thermal cycling block, incubate the reaction mixtures for 5 min at 94°C.
4. Add 2 μl of Vent DNA Polymerase (final concentration 2 Units).
5. Randomly join the fragments in a thermal cycling block as follows:
94°C for 1 min
65°C for 1 min
72°C for 2 min
6. Maintain the temperature of the thermal cycling block at 94°C. Add 1 μl of each set of Flanking Primers (see Hint #2 and Section B) to each microcentrifuge tube (reaction and control mixtures).
7. Amplify the fragments in a thermal cycling block as follows:
94°C for 1 min
60°C for 1 min
72°C for 1 min
B. Polymerase Chain Reaction Product Purification
1. Analyze 3 μl of the PCR reaction products on a 1% Agarose Gel (see Protocol ID#1265) to determine splicing success (see Hint #3).
2. Purify the spliced gene repertoires by Agarose Gel Electrophoresis on a 1.0% Agarose Gel (see Protocol ID#1265).
3. Extract the PCR products from the gel. Use Protocol ID#122 or Geneclean™ (Bio 101, Inc also see Hint #4 and Hint #5).
4. Resuspend the recovered DNA fragments in 20 μl of ddH2O.
5. Determine the DNA concentration by Electrophoresis through a 1% Agarose Gel using DNA markers of known size and concentration (see Protocol ID#1265).
C. PCR Primers
scFv Flanking Primers (see Complete Primer Sequences).
Vent Polymerase Buffer (10X) 100 mM KCl
New England Biolabs buffer supplied with Vent Polymerase can also be used
1 % Triton X-100
20 mM MgSO4
100 mM Ammonium Sulfate ((NH4)2SO4)
200 mM Tris-HCl, pH 8.8 at 25°C
dNTP Mix (20X) 5 mM dCTP
5 mM dGTP
5 mM dATP
5 mM dTTP
BioReagents and Chemicals
DNA Polymerase, Vent
1. Prepare two sets of Reaction Tubes, one containing the Vκ repertoire DNA and the other containing the Vγ repertoire DNA.
2. Add an equimolar mixture of the 6 VHBACK primers (listed in Section B) and add an equimolar mixture of the 5 JκFOR and 4JλFOR primers (listed in Section B). The final total concentration of each primer mix is 10 pmol/μl.
3. The spliced scFv product should be between 0.8 to 0.9 kb. There should not be a product of this size in the two negative control reactions.
4. The contributor of this protocol suggests using the Geneclean™ Kit. Follow the manufacturer's instructions to isolate the DNA. If using Protocol ID#122, substitute ddH2O for TE Buffer.
5. For library creation, the spliced scFv gene repertoires must be reamplified to append restriction sites (see Protocol ID#2199).
6. In Two Fragment PCR assembly methods, V-regions are amplified with regions of the primers that overlap in such a way that mixing the two V-regions recreates a linker region joining the two V-genes. This is only possible if the linker region is not repetitive, and therefore is not suitable for the repetitive gly-ser linker. When using the gly-ser linker, the region of overlap must extend to the opposite chain; or the linker should be amplified as a series of separate fragments, each of which has homology to a different VH and VL chain gene (see Image #1). This is termed 3-fragment assembly. In both cases, a number of amplification cycles without the addition of external primers are first performed. These involve an initial annealing of the regions of overlap followed by an extension. In this way, VH regions are joined to VL regions via the linker, and the complete scFv can then be amplified by subsequent PCR in which external primers containing suitable restriction enzyme recognition sites are added. The reaction is easier to perform if separate VH and VL libraries are first made and reamplified to create assembly fragments. Restriction enzymes that do not cut frequently within V regions must be used. AscI, BssHII, BstBI, MluI, NotI, PmlI, SalI, SfiI, SnaBI, SrfI are all suitable, cutting no more than one germline V-gene.
Citation and/or Web Resources
1. Phage Display: A Practical Approach. Eds. T. Clackson and H. Lowman. Oxford University Press, (2001) in press.