Lecture 23 - DNA techniques Flashcards
What are the 7 different recombinant DNA techniques
DNA purification DNA cloning PCR Site-directed mutagenesis DNA libraries DNA sequencing Genome projects
Describe DNA purification
Extraction of DNA.
Lyse cells, centrifuge to remove debris, remove protein, then concentrate DNA and treat with ribonucleases to remove RNA
Describe the 2 different cloning vectors
Plasmids and bacteriophages (lambda)
Plasmid: circular DNA with restriction endonuclease sites to open up vector and insert DNA
Bacteriophage: introduce DNA into E.coli, replace some of the non-functional parts of the bacteriophage genome with similar-sized foreign DNA segments to ensure that the genome will get packaged into the protein coat
How does cloning work?
Cut the vector DNA at RE sties, excise the gene with the same REs (or generate gene through PCR), create recombinant DNA. Insert DNA into host cell, select host cells with recombinant DNA
How do restriction enzymes work?
hydrolyze phosphodiester bond –> creates blunt ends or sticky ends. Sticky ends can anneal and ligate to other complementary sequences, blunt ends can’t ligate as efficiently because they don’t anneal (base-pair)
What do DNA ligases do?
form phosphodiester bonds
How do you select for host cells containing the vector?
Use inactivation of lacZ gene to check if vector took up gene. LacZ codes for beta-galactosidase, which cleaves X-gal to create a blue product. Use plasmid with an RE in the lacZ site, so that foreign gene is inserted into lacZ which inactivates it. Therefore, blue product doesn’t form, so you select for colourless cells. Can select for host cells that took up vector through antiobiotics. Have vector code for antibiotic resistance, and then grow in medium containing antibiotics, only host cells that take up vector will have conferred resistance.
How do you overexpress the product?
Tag a gene with a epitope tag, use the epitope tag on the protein to synthesize and precipitate the tagged protein
How do you modify a gene?
Site-directed mutagenesis
2 methods
1) cut out a section from the plasmid and replace with synthetic sequence
2) place short synthetic oligonucleotide on ssDNA that serves as a primer, then replication occurs.
How can DNA be sequenced?
Sanger or automated Sanger sequencing
Sanger: 4 tubes, each tube has deoxynucleotides and one dideoxynucleotide that halts replication. Because you know which is which, with enough replication cycles, will have segments that end at every position. Then run through gel electrophoresis to identify. Will receive complementary sequence to gene.
Automated Sanger: same, except only one tube and each dideoxynucleotide contains a radioactive tag to identify it
What is PCR used for and how is it done?
amplify specific DNA sequences
Heat to separate strands, add primers, then add DNA pol and dNTPs
Used for forensics, detect rare sequences, study evolution, INTRODUCE RE sites to vectors through primers containing RE sites
