Restriction Enzymes & Gel Electrophoresis 2024 PDF

14. Restriction enzymes / restriction analysis BIOC3400 Archibald—L14 1 Chapter 7, 211-215 Restriction endonucleases (restriction enzymes) Enzymes that catalyze the double-strand cleavage of DNA at specific or non-specific base sequenc...

14. Restriction enzymes / restriction analysis BIOC3400 Archibald—L14 1 Chapter 7, 211-215 Restriction endonucleases (restriction enzymes) Enzymes that catalyze the double-strand cleavage of DNA at specific or non-specific base sequences. Name comes from “Restriction—modification” Many bacteria use site-specific methylation to mark their own DNA and DNA cleavage to inactivate foreign DNA (e.g., viral DNA) Strain-specific restriction enzymes and methylases, work in pairs Three types: I, II and III (I and III are very similar; we will focus on type II enzymes) tangy double se a ↓ marks dearage Type I Methylase and endonuclease activity in one enzyme Cleavage site outside of recognition site in non-specific sequence (up to 10 kilobases away from recognition site) Cleavage requires ATP EcoK recognition sequence: m 5’-AACNNNNNNGTGC-3’ m = methylation site 3’-TTGNNNNNNCACG-5’ m  BIOC3400 Archibald—L14 2 Restriction enzymes Type II Endonuclease activity only (methylase is another enzyme) Cut sites within recognition site ATP-independent EcoR1 recognition sequence and cleavage site: m 5’-GAATTC-3’ · m = methylation site 3’-CTTAAG-5’ · = cleavage site m Different restriction enzymes can generate different types of ends: · “Sticky” or “cohesive” ends -3’ overhang -5’ overhang O so “Blunt” or “flush” ends  BIOC3400 Archibald—L14 3 Restriction enzymes Summary, characteristics of type II restriction enzymes * base-cutters larger are rate *NOTE We will focus only on the Type II restriction enzymes  Restriction enzymes BIOC3400 Archibald—L14 4 Recognition sites—are often palindromes with two-fold symmetry about a central axis Different restriction endonucleases can produce compatible ends: - symmeting line & palindromes I EcoR1 homodimer bound to DNA ↳hammer Isoschizomers: Restriction enzymes that recognize the same DNA sequence, but may or may not cleave at the same position e.g., SacI and SstI both recognize and cleave in the same position: 5’-GAGCT/C-3’ HhaI an HinP1I recognize same sequence, but cleave at different positions: HhaI 5’-GCG/C-3’ HinP1I 5’-G/CGC-3’ Restriction enzymes BIOC3400 Archibald—L14 5 Restriction enzymes can be used to create recombinant molecules (see Lecture 15)  Principles of gel electrophoresis BIOC3400 Archibald—L14 6 Electrophoresis The movement of charged molecules in an electric field, with negatively-charged molecules moving towards the positive electrode, and positively-charged ones towards the negative electrode. Gel electrophoresis A WIDELY used method for separating DNA (or RNA) molecules based on differences in their lengths. Separation medium is a gel. Gel electrophoresis is very useful for analyzing DNA because: DNA has one unit charge / residue, its charge is proportional to its length. The molecular sieving effect determines the relative mobility of DNA molecules at a given gel concentration, and this effect is proportional to the length of the molecule. Therefore, electrophoresis can be used to separate DNA fragments on the basis of size alone. Mobility of linear DNA fragments is inversely proportional to the logarithm of their molecular weight.  Gel electrophoresis BIOC3400 Archibald—L14 7 Used for: Size analysis of restriction fragments Restriction mapping Purification of DNA fragments to be cloned or sequenced DNA sequencing Two types of gel are used in molecular biology: Basic Methods in Molecular Biology, 2nd Ed. Agarose A polysaccharide that forms gels with pores 100-300 nm, depending on concentration of agarose used useful for resolving fragments between ~100 and 50,000 nucleotides in length Polyacrylamide A cross-linked polymer, used for electrophoretic analysis of proteins and small nucleic acids Can be used to resolve fragments that differ by as little as one nucleotide in length  Restriction analysis BIOC3400 Archibald—L14 8 Restriction enzyme mapping Digest a DNA molecule with different restriction enzymes with different target sequences and compare the fragment sizes. The result is a ‘restriction map’ NOTE: Restriction maps are easy to generate if there are relatively few cut sites for the enzymes being used Restriction mapping is generally only feasible for small molecules (although rare base cutters (e.g., NotI) make it possible to generate maps for larger fragments) Example: digest DNA fragment with two different enzymes, individually and together (i.e., single and double digests)  BIOC3400 Archibald—L14 9 DNA Strider Software for DNA sequence analysis Restriction enzyme cut site identification  BIOC3400 Archibald—L14 10 DNA Strider Software for DNA sequence analysis Identifying potential protein-coding regions in DNA Open reading frame (ORF) maps 6-frame translation Start codons = short ticks Stop codons = long ticks ORF maps are very useful for scanning a new DNA sequence for potential coding capacity. Might it contain a gene? One can look for long, uninterrupted stretches of DNA with a start codon at the beginning and a stop at the end.