Lecture 23: Technology
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Recombinant DNA: The Dawn of Molecular Biology What is recombinant DNA?
DNA formed artificially by combining constituents from different organisms
Recombinant DNA: The Dawn of Molecular Biology What is recombinant DNA created from?
Restriction endonuclease digested genomes, ligated into plasmids
Recombinant DNA: The Dawn of Molecular Biology How is DNA cleaved?
DNA is cleaved at specific sequences by restriction endonucleases - They often bind and cut the phosphodiester backbone at palindromic sequences
Recombinant DNA: The Dawn of Molecular Biology What happens if restriction endonucleases cut the DNA and leave overhangs?
Sticky ends
Recombinant DNA: The Dawn of Molecular Biology What happens when enzymes cut at the same site on both strands?
Blunt ends
Recombinant DNA: The Dawn of Molecular Biology What is ligation?
Joining fragments together
Recombinant DNA: The Dawn of Molecular Biology What are plasmids?
Very small circular DNA w few genes - Selected for via ABX resistance markers
Recombinant DNA: The Dawn of Molecular Biology Describe the relationship between DNA, endonucleases, DNA ligase, and plasmids
Endonucleases cut a piece of DNA The fragment gets pasted into a plasmid and ligated with DNA ligase
Recombinant DNA: The Dawn of Molecular Biology What is bacterial transformation?
Bacteria take up foreign DNA from environment
Recombinant DNA: The Dawn of Molecular Biology What marker do plasmids contain?
They contain a selection markers
Recombinant DNA: The Dawn of Molecular Biology What are selection markers?
A specific gene in a plasmid that allow for the identification and selection of cells that have successfully taken up the plasmid
Recombinant DNA: The Dawn of Molecular Biology Describe the relationship between recombination DNA, a host cell and a foreign cell
Uptaken DNA can recombine with the host’s chromosomal DNA - The host incoperates the new DNA into its cell machinery - Can lead to genetic changes such as antibiotic resistance
Recombinant DNA: The Dawn of Molecular Biology What genes are some selection markers?
Genes that confer antibiotic resistance
Recombinant DNA: The Dawn of Molecular Biology What bacterial cells will grow into a medium containing the corresponding antibiotic?
Bacterial cells transformed with the plasmid containing an antibiotic resistance gene will grow in a medium containing the corresponding antibiotic
Recombinant DNA: The Dawn of Molecular Biology What cells will survive and grow in terms of antibiotic resistance?
Cells that have successfully taken ip the plasmid
PCR What does PCR stand for?
Polymerase Chain Reaction
PCR What is the goal of PCR?
Amplification of a region of target DNA
PCR What are the steps for PCR?
1) Heat DNA to seperate strands 2) Add synthetic DNA oligonucleotide primers 3) Add Taq polymerase to catalyze 5’ -> 3’ DNA synthesis
PCR What can you do after you have multiple copies of DNA made from PCR?
After you have multiple copies you can cut with restriction enzymes to insert into plasmid
Argose Gel Electrophoresis What is the goal of Gel electrophoresis?
To determine the size of fragments
Argose Gel Electrophoresis What are the steps of Gel electrophoresis?
1) Load DNA into wells at one end of argose gel 2) Connect to power supply - Cathode (negative charge) = at the same end of the gel as the wells - Anode (positive charge) = at the opposite end of the gel from the wells 3) DNA moves toward anode and movement speed/final positions depend on size
Argose Gel Electrophoresis How is DNA normally charged?
Negatively
Argose Gel Electrophoresis Are the longer molecules at the longer vs. shorter molecules in the Electrophoresis plate?
Longer = at the top Shorter = at the bottom
Cloning the sequence of genes DNA contains all introns and exons How can you create recombinant DNA with just exons?
Complementary DNA (cDNA)