Lecture 16

Lecture 16 - LS3 MOLECULAR BI OLOGY LECTURE 15 Overview Restriction Enzymes& DNA Mapping Dr Randolph Wall Pairs of enzymes that recognize

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Unformatted text preview: LS3 MOLECULAR BI OLOGY LECTURE 15 Overview Restriction Enzymes & DNA Mapping Dr. Randolph Wall Pairs of enzymes that recognize specific 4 - 8 bp DNA sequences (usually palindromes, identical inverted 5’ -> 3’ sequences on both DNA strands) • The modification enzyme (methylase) adds methyl groups to nucleotides in this specific sequence on both DNA strands - such methylated DNA is resistent to cutting by the restriction enzyme (a.k.a. restriction endonuclease) • The restriction enzyme will cleave any DNA that is not methylated at this specific recognition sequence • Foreign DNAs (e.g., phage DNA) are not methylated • The restriction enzyme cleaves the foreign DNA at the restriction sites, rendering it susceptible to further digestion by other nucleases (exonucleases) • Hence, restriction/ modification systems function in nature to protect bacteria from viruses and other foreign DNAs Discovery of a Primative I mmune System: Restriction/ Modification Systems in Prokaryotes GAATTC CTTAAG 5’ 3’ 3’ 5’ CH 3 CH 3 EcoRl restriction N O CLEAVAGE Methylated DNA is Resistant to Restriction Enzyme Cleavage, Unmethylated DNA is cut EcoRl site v v Most restriction sites have short inverted repeat sequences (palindromes) Blunt ends Sticky ends 5’ overhang 5’ overhang 3’ overhang Selected Restriction Enzymes & Cutting Sites enzyme recognition sequence (5’->3’) type of ends Alul AG v CT blunt Haelll GG v CC “ Hpall C v CGG 5’ overhang Sau3A1 v GATC “ BamHl G v GATCC “ Bgl ll A v GATCT “ EcoRl G v AATTC “ Hindlll A v AGCTT “ Pstl CTGCA v G 3’ overhang Sacl GAGCT v C “ Sall G v TCGAC 5’ overhang Smal* CCC v GGG blunt Xmal* C v CCGGG 5’ overhang * isoschizomers - same recognition sequences Some Restriction Endonucleases and Their Recognition Sequences Enzyme Sequence Cut Frequency * Sau3A1 5’- GATC-3’ 0.25 kb EcoRl 5’-G AATTC-3’ 4 kb Notl 5’-GC GGCCGC-3’ 65 kb * Frequency = 1/ 4 n , where n = the number of bp in the recognition sequence: 4 4 = 256 bp, 4 6 = 4096 bp, 4 8 = ~65,000 bp Approximate v v v Plasmids • Circular, double-stranded DNA (sizes 1 - 200 kb) • Occur naturally in bacteria, yeast, higher eukaryotes • Exist in a parasitic or symbiotic relationship within their host cells • Replicate separately from host cell’s chromosomal DNA due to presence of plasmid DNA replication origin (ori) • Contain ancillary genes useful to plasmid host cells (e.g., genes encoding toxins, antibiotic resistence, heavy metal resistence, restriction/ modification enzymes) A plasmid constructed for use as a vector in recombinant DNA cloning requires an ori, a selectable marker and one or more unique cloning sites - small size (~2-3kb) also useful. I ntroduction of recombinant DNA-vectors into host cells is called transformation (a.k.a. transfection) Versatile Plasmid Cloning Vector Containing Polylinker with Multiple Unique Restriction Sites for I nserting DNA Fragments...
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This note was uploaded on 07/02/2008 for the course LS 3 taught by Professor Lin during the Spring '06 term at UCLA.

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Lecture 16 - LS3 MOLECULAR BI OLOGY LECTURE 15 Overview Restriction Enzymes& DNA Mapping Dr Randolph Wall Pairs of enzymes that recognize

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