PCR
To manipulate gene products obtained from gene-bank, our team used PCR/Real-Time PCR methods. Commercially obtained genes were containing few more base paires added at front and back site. As FGPR, the receptor protein of human, has human-specific signal peptide at its starting point, this region had to be replaced
by S.pombe specific signal peptide to express the protein at membrane region. To do this, PCR primer containing signal peptide region of S.pombe were synthesized with method described below. Then, FGPR gene was amplifed by PCR to get gene product which its signal region is replaced. Also, additional sequence at rear site of gene was
removed. STAT gene passed through similar process; removing additional region.
PCR was done by commercial service sponsered by Bioneer.Inc (Korea), using AccuPower PCR system supplied by same company as reference below.
Oligo Synthesis
We were supported our oligo-synthesis by Bioneer.
We requested about fusion antibody gene that is mainly used in our project.
Process about oligo-synthesis by Bioneer is summarized at below.
Codon optimization Oligo Design S/W
-> High-efficient optimiztion software made Bioinformatics technology of KAIST
Mega-base Oligo Synthesizer
-> Mass oligo synthesizer which can synthesis 25 million base pair of oligonucleotide
In addition, through the following processes, Bioneer completed our oligo-synthesis perfectly.
Sequencing Service
-> High-accuracy sequencing service
AccuRapid Cell-Free Protein Expression Kit
-> Cell free protein expression system
Gene Synthesis Kit
-> World-first one-stop gene synthesis kit
Homologous Recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks. Homologous recombination also produces new combinations of DNA sequences during meiosis, the process by which eukaryotes like animals and many plants make sperm and egg cells. These new combinations of DNA represent genetic variation in offspring, which in turn enables populations to adapt during the course of evolution. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.
We use this technique at yeast S. pombe. Diploid deletion mutants in the S. pombe genome were systematically constructed with targeted mutagenesis at each target ORF. The chromosomal location of the ORFs and their DNA sequence information were obtained from the public fission yeast database at the Welcome Trust Sanger Institute. The deletion cassette module construct contains a selection marker (Fig. 1), tag sequences (molecular barcodes), and the sequences for homologous recombination (Fig 2). The deletion cassette modules were constructed by PCR with well-designed primers. The cassettes were transformed into the S. pombe, SP286 diploid host strain (h+/ h+, ade6-M210/ade6-M216 ura4-D18/ura4-D18 leu1-32/leu1-32). Deletion of the target ORF was screened for by G418 antibiotic selection (Fig 3). If chromosomal integration occurs properly via homologous recombination at the target ORF in a colony, that colony would obtain antibiotic resistance from the KanMX4 selection marker gene.
References
Bioneer PCR service:
http://www.bioneer.co.kr/product/product_sub.jsp?pclassCode=1
Bioneer Oligo sythetis service
Put reference list here
|
"
