Restriction Endonucleases, RFLP, and Gene Cloning

Restriction endonucleases, RFLP, and gene cloning Endonucleases Enzymes that degrade DNA within the molecule rather than from either end (exonucleases) Restriction endonucleases: Enzymes that recognize and cut (break) the phosphodiester bond between nucleotides at specific sequences (4- to 8-bp restriction sites) generating restriction fragments. Type II restriction endonucleases: Always cleave always at the same place generating the same set of fragments EcoRI (isolated from E. coli) cuts at 5'-GAATTC-3‘ Some enzymes cut DNA at related sites HinfI (from Haemophilus influenzae) recognizes 5'-ANTC-3' ('N' is any nucleotide) Cuts at 5'-AATC-3', 5‘-ATTC-3', 5‘-AGTC-3' and 5'-ACTC-3’ Biological purpose of restriction endonucleases They are present in bacteria to protect them from bacteriophages that infect bacteria by transferring their DNA into them restricting their growth. Types of cleavages Restriction enzymes cut DNA in two different ways: Blunt: enzymes cut at the same position on both strands giving a blunt ended fragments Staggered (off-center): enzymes cut the two DNA strands at different positions generating sticky or cohesive ends The DNA fragments have short single-stranded overhangs at each end. 5’ vs. 3’ overhangs Palindromic sequence The sequences recognized by restriction endonucleases—their sites of action—read the same from left to right as they do from right to left (on the complementary strand). DNA ligase Covalently joins DNA ends (example, restriction fragments) Catalyzing the formation of 3’→ 5’ phosphodiester bonds between the 3-hydroxyl end of one strand and the 5-phosphate end of another strand Advantage of restriction endonucleases Restriction fragment length polymorphism (RFLP) Cloning DNA polymorphisms Individual variations in DNA sequence may create or remove restriction-enzyme recognition sites generating different restriction fragments Remember: our cells are diploid (alleles can be homozygous or heterozygous) What is an allele? 38 39 Restriction fragment length polymorphism The presence of different DNA forms in individuals generates a restriction fragment length polymorphism, or RFLP. These can be detected by Gel electrophoresis Southern blotting Example RFLP in the clinic RFLP can be used as diagnostic tools. For example, if a mutation that results in the development of a disease also causes the generation of distinctive RFLP fragments, then we can tell if the person is diseased as a result of this mutation from which parent this allele is inherited 42 Disease detection by RFLP Think!! What would you see in a gel? 45 Example 1: Disease detection by RFLP (sickle cell anemia) 46 Normal Diseases Normal/ carrier Father Mother Son1 Son2 Son3 Example 2: Disease detection by ASO (Cystic fibrosis) ASO: Allele-specific oligonucleotide 48 Example 3: Paternity testing 49 Example 4: Forensics 50 Cloning Cloning means that you make several copies of one thing. A clone is a genetically identical population, whether of organisms, cells, viruses, or DNA molecules. Every member of the population is derived from a single cell, virus, or DNA molecule. How do we clone a DNA molecule? a DNA fragment of interest is inserted into a DNA carrier (called a vector) that can be replicated. The resulting DNA molecule is what is known as a recombinant DNA molecule. Using plasmids as vectors Bacterial plasmids are considered excellent vectors. These are bacterial circular DNA that is not part of the main circular DNA chromosome of the bacterium. A plasmid exists as a closed circle and replicates independently of the main bacterial genome. Features of plasmids Most plasmid vectors contain at least three essential parts required for DNA cloning: Can replicate Can be selected for/against by an internal drugresistance gene (selectable marker) Can inset a foreign DNA fragment Making of recombinant DNA Both DNA fragments (the DNA to be cloned and a vector) are cut by the same restriction endonuclease that makes sticky-ended DNA fragments When mixed, they will bind to each other 55 56 Cell clones Cloned DNA 57