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DNA Cloning PDF
DNA Cloning PDF
cleave with RE
insert DNA with
DNA ligase
Basic steps in DNA cloning
• Identify and isolate specific DNA
• Antibiotic resistance
• “Blue-white” screening
Cloning of human genes
• Factor VIII - hemophilia
• Insulin - diabetes
Genomic DNA library
• Coding sequences
• Intronic sequences
Step 2 Anneal
Step 3 Extend
PCR - Advantages
• Speed
• Ease of use
• Sensitive
• Robust
PCR - Disadvantages
• Need sequence information
• Site-directed mutagenesis
• Animal models
Normal gene
Replacement Addition
Knockout
• Gene replacement - info about the
mutant gene