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Historical Perspective • Pre-history before 1928 • Ancient before 1944 • Medieval 1944-1952 • Renaissance 1953-1971 • Modern Era 1971 to present
Further Historical Perspective Geneticists have known for a long time how to isolate DNA from cells. Geneticists have known for a long time how to chop DNA into small pieces. What geneticists did not know how to do until the early 1970s was to replicate small fragments of DNA.
1970s Breakthrough: • The discovery of the restriction enzyme (or restriction endonuclease).
Properties of RE: • Cut double-stranded DNA at specific target sites. • Allow fragments of DNA that have been cut with the same RE to be rejoined.
Properties of RE con’t: • There are hundreds of popular RE. They all recognize a small target sequence (4-8 b.p.).
The joining of two DNA fragments by DNA ligase produces a recombinant DNA molecule
Therefore, eukaryotic DNA could be propagated in prokaryotic cells.A great breakthrough!!!!!
Carriers of foreign DNA are Vectors: • Most vectors are derived from: • 1.Plasmids • 2. Bacteriophages • 3. Cosmids (artificial constructions)
A prokaryotic vector should: • 1. Be capable of autonomous replication independent of the main bacterial chromosome • 2. Be easy to isolate, i.e. small. • 3. Be non-toxic to host cells. • 4. Have space for foreign inserts. • 5. Have unique restriction sites for common restriction enzymes. • 6. Have convenient markers for selection of transformants, e.g. antibiotic resistance genes. • 7. Be relaxed, i.e. multiple copies in a host cell.
Introduction to PCR • PCR (polymerase chain reaction). * p r c
What is PCR? • PCR is site-specific in vitro DNA replication.
DNA Replication Review: • Add DNA polymerase, all 4 DNA building blocks ??? 5’ CTGACGCTGCTGCATGCTAGCT 3’ 3’ GACTACGACGACGTACGATCGA 5’
DNA Replication Review: • Primers are required: 5’ CTGACGCTGCTGCATGCTAGCT 3’ CGA 5’ 5’ CTG 3’ GACTACGACGACGTACGATCGA 5’
DNA Replication Review: • Primers are required: 5’ CTGACGCTGCTGCATGCTAGCT 3’ . . . t a c g a t CGA 5’ 5’ CTG a t g c t g . . . . 3’ GACTACGACGACGTACGATCGA 5’
Action of DNA Polymerases: • DNA polymerases can add new nucleotides to an exposed 3’ end!
PCR reaction mix: • All 4 DNA building blocks (A,C, G, & T) • Taq DNA polymerase (heat resistant) • DNA to be replicated • A pair of primers
Human Chromosome # 8: | | | | | || || || || | add primers: | | | || || || || | Human Chromosome # 8: | | | || || ||| || | | | || || || || |
PCR product? • The binding of the primers determines where the DNA is replicated. • The PCR product is a double-stranded DNA molecule with its ends defined by the location of primer binding sites.
PCR • PCR reaction mix incubates for about 2 hours in a thermocycler (fancy incubator). • The thermocycler heats and cools through 30-40 temperatures cycles.
Two impt. PCR questions: • What is the source of a pair of primers? What information is a prerequisite for PCR?
PCR Applications: #1- Cell-free rapid gene cloning!! #2- Gene cloning/amplification from a miniscule sample size.
After completion of the run, add a DNA staining material and visualize the DNA under UV light.