Quality control of genomic DNA libraries using PCR
Quality control of genomic DNA libraries using PCR
In this scenario, human genomic DNA is treated with a mixture of restriction endonucleases (the recognition site of the endonuclease mixture is 4bp) to produce fragments of roughly 50 to 500bp. The ultimate use of this type of expression library is the isolation of gene fragments encoding functional polypeptide or protein fragments by utilizing phenotypic and biochemical screening methods. After treatment with KlenowDNA polymerase, the genomic DNA fragments become flat-ended and are ligated into a vector linearized with Pml Ⅰ (an enzyme that produces a flat-ended product), then transformed into bacteria and amplified to obtain the library. This experiment is from PCR Laboratory Guide (2nd edition) by Seed Kang and Qu Lijia.
Operation method
Quality control of genomic DNA libraries using PCR
Materials and Instruments
ddH20 Vent buffer Vent DNA polymerase dNTP plasmid DNA PCR primers Move I. Materials For more product details, please visit Aladdin Scientific website.
Reagents and equipment for agarose gel electrophoresis Thermocycler
1. Buffers, solutions and reagents
ddH20
2. enzymes and enzyme buffers
10X Vent buffer
Vent DNA Polymerase
3. Nucleic acids and oligonucleotides
dNTP, 20 mmol/L each
PCR primers that anneal to vector sequences flanking genomic DNA library cloning sites
Plasmid DNA (see step 1 below)
4. Specialized equipment
Reagents and equipment for agarose gel electrophoresis, including ethidium bromide (see step 5)
Thermocycler
II. Methods
1. After amplification in bacteria, set up the following reactions with plasmid DNA prepared from a genomic library.
10~50ng of plasmid DNA
50pmol of each primer
5ul of 10XVent buffer
dATP, dCTP, dTTP and dGTP, 0.5ul each
1U of Vent DNA Polymerase
Vent DNA Polymerase, 0.5ul of each of dATP, dCTP, dTTP and dGTP.
Separate reactions should be performed for each of the following plasmid vectors:
Expression vector without library DNA insertion
Genomic DNA library mixture
Small amount of DNA, prepared from a single bacterial clone of the genomic DNA library
2. Preheat the mixture in a thermal cycler at 94°C for 20s.
3. Perform 30 rounds of PCR cycles according to the following parameters.
94°C15s
55°C10s (or appropriate annealing temperature with primers)
72°C40s
Extend the extension time of the last cycle to 60s.
4. Cool to 4C.
5. Take 25ul of each reaction product, run 4% agarose gel electrophoresis and analyze with ethidium bromide staining (Sambrook and Russell 2001)
III.
In the example shown in Fig. 26-6, PCR analysis of the library mixture, in which the primers used are sequences flanking the PmlⅠ locus, shows that the library contains insert fragments of the target size, but there is a large amount of contamination of the original vector [Fig. 26-6(a), lane 2]. PCR analysis of a single clone selected from the library verified the result of using the library mixture as a template and showed that the original vector in the library accounted for about 40% of the total [10 out of 24 clones analyzed, Fig. 26-6(a), lanes 3~26]. To improve the quality of the libraries, the libraries were digested with Pml I enzyme and then re-amplified in bacteria to minimize contamination of the original vector. PCR analysis of the library mixture showed that the contamination of the uninserted looped vector was greatly reduced after digestion with PmlⅠ [Fig. 26-6(b), lane 2 and lane 3]. After digestion with PmlⅠ and re-amplification in bacteria, PCR analysis of single clones randomly selected from the library confirmed that the frequency of insert-free DNA was much lower than that of the original library [0 out of 20 clones, less than 5%, Fig. 26-6(b), lanes 4~23] and that the clones contained the expected size of the insert fragments. DNA sequence analysis of the clones in the genomic fragment library showed that the library contained human genomic DNA fragments of the expected size (data not shown). Thus, PCR provides a rapid, sensitive, and convenient method to analyze both raw and improved libraries. 