Preparation of double-stranded DNA libraries encoding randomized peptides by PCR
Preparation of double-stranded DNA libraries encoding randomized peptides by PCR
PCR serves two important purposes when constructing libraries using synthetic oligonucleotides. First, by amplifying the library with primers that anneal to specific flanking sequences, a large number of PCR products can be generated using only a small number of different templates. Second, PCR synthesis is an efficient method for generating complex complementary strands of library DNA. This experiment is based on the PCR Laboratory Guide (2nd ed.) by Kang Seed and Lijia Qu.
Operation method
Preparation of double-stranded DNA libraries encoding randomized peptides by PCR
Materials and Instruments
Restriction endonucleases and buffers Sterilized double-distilled water Tris-HCl Biotin-labeled PCR primers Move I. Materials For more product details, please visit Aladdin Scientific website.
Streptavidin anti-biotin protein magnetic beads Reagents and equipment required for agarose gel electrophoresis
1. Buffers, solutions and reagents
Sterilized double distilled water ( ddH20 )
10 mmol/L Tris-HCl, pH 8.0
2. Enzyme and enzyme buffer
Restriction endonuclease and buffer (see step 9)
10XVent buffer
VentDNA Polymerase
3. Nucleic acids and oligonucleotides
Biotin-labeled PCR primers, at least 15 bp in length, with biotin modification at the 5' end (see Figure 26-1). 
NNK library encoding 12 amino acids. The library design is shown in Figure 26-1(a).
The synthesized strands contain a library of random polypeptides with a specific flanking sequence added to both their 3' and 5' ends to serve as primer binding sites and contain restriction endonuclease recognition sites to facilitate cloning of the library DNA into a suitable vector system. The flanking sequences should be at least 15 bp in length and should be designed with 3 criteria: 1. they should be valid PCR primer binding sites, 2. they should contain restriction sites for subsequent cloning steps (optional: restriction sites can be located in the overlap region between the 5' end of the PCR primer and the library oligonucleotide and not inside the library oligonucleotide), and 3. the restriction sites should be located at the 5' end of the PCR primer and inside the library oligonucleotide. Because one or both ends of the penwing DNA sequence are likely to represent amino acids in the peptide library, they should not contain codons that affect library function. During synthesis of the coding region of the library, the first two sites of each codon contain equal amounts of the four nucleotides, whereas at the third site the ratio of G+T is 1:1 [Fig. 26-1(a)]. The absence of A and C nucleotides in the third site reduces the possibility of a stop codon, but still allows the encoding of 20 amino acids.
dNTP (20 mmol/L each)
4. other
Reagents and equipment for agarose gel electrophoresis, including ethidium bromide (see step 11)
QIAprep Miniprep silica column (QIAGEN)
Streptavidin anti-biotin protein magnetic beads (Dynal)
II. Methods
1. First round of polymerization reaction: PCR amplification of the library
(1) Perform the following reactions to amplify single-stranded synthetic oligonucleotides into efficient starting material for library construction. The amounts given below are for a 100ul system. To construct a large-scale library, prepare 1 ml of reactants and divide them equally into 10 separate PCR tubes.
10pmol of library oligonucleotide
2U of Vent DNA polymerase
Biotin-labeled PCR primers, 100pmol each
10ul of 10XVent buffer
dATP, dCTP, dTTP and dGTP, 1ul each
Dissolve with ddH2O to 100ul
(2) Preheat the mixture for 20s at 94°C in a thermal cycler.
(3) Perform 30 rounds of PCR cycling according to the following parameters.
94°C10s
55°C10s (or appropriate annealing temperature with primers)
72°C15s
(4) Cool to 4°C.
2. Second round of polymerization: synthesize paired double-stranded library DNA.
(5) Add 10ul of 10X reaction buffer, 100pmol of each primer, and 2U of Vent DNA polymerase to every 100ul of the first reaction product.
(6) Perform a single round of cycling: 94°C for 30s, 55°C for 30s, 72°C for 15s.
(7) Cool to 4°C.
(8) Purify the second round of amplification products with a column of QIAprep and elute with 10 mmol/L Tris (pH 8.0).
(9) Digest the eluted product with a suitable endonuclease (Fig. 26-2, lane 1), then precipitate the DNA with ethanol, and dissolve with ddH2O (Sambrook and Russell 2001). As a result of this digestion reaction, biotin-labeled DNA ends will be released from the core of the library.
(10) Remove the biotin-labeled DNA ends with streptavidin anti-biotin protein magnetic beads (Korn et al. 1992). This step will increase the yield of the target ligation product because these ends are able to be ligated to the library insert or expression vector. The Streptavidin affinity step also removes any PCR products that are not cleaved or partially cleaved (Figure 26-2, lanes 3 and 4).
(11) Analyze the reaction intermediate and final products on a 4% agarose gel. The final product (Figure 26-2, lane 4) is a high-purity and highly complex double-stranded library DNA with prominent ends that can be ligated directly into a suitable expression system. 