First Prelim Thursday February 18 in class Material included - up to today?s lecture A - Mariano go to PLS 233 McConnell - Z go to RRB125 Reviews on Monday 4:30 - 6:00 pm Warren 245 Wednesday 5:00 - 6:30 pm Warren 131 Restriction endonucleases Discoverers - Daniel Nathans, Hamilton Smith and Arber Werner Shared Nobel Prize in Medicine in 1978 Blunt ends 5? overhang 5? 3? overhang 3? The number of sites on a given DNA molecule cleaved by a particular restriction enzyme depends on: 1. [A+T]/[G+C] content of the DNA molecule 2. The recognition sequence of the restriction endonuclease For example: EcoRI has a specific recognition sequence of 6 bp GAATTC. If the [A+T]/[G+C] content of a DNA molecule is 1, then the probability of the sequence GAATTC occurring is: 1/4x1/4x1/4x1/4x1/4x1/4 = 1/46 =1/4096 What is the average size of the restriction fragments generated by the enzyme Not1 from the genome of a primate whose DNA has an [A+T]/[G+C] content of 3/2 (40%[G+C])? How many times will NotI cut the genome which is estimated to contain 3x109 bp? Not site is GCGGCCGC G/[A+T+G+C] = 1/5 Frequency of Not sites = 1/58 = 1/390625 Cleaves 7680 times Answer: Problems - Ch.9: 1, 2, 3, 4, 9 Recombinant DNA Cloning large DNA segments for genome sequencing Assembling the sequences DNA sequencing Some of the utilities of restriction endonucleases ---TACGAATTCAGC--- ---ATGCTTAAGTCG--- ---CCTGAATTCGAA--- ---GGACTTAAGCTT--- ---CCTG AATTCGAA--- ---GGACTTAA GCTT--- ---TACG AATTCAGC--- ---ATGCTTAA GTCG--- ---TACGAATTCGAA--- ---ATGCTTAAGCTT--- DNA ligase (E. coli or T4 ligase will ligate nicks with 3?OH and 5?P) EcoRI EcoRI E. coli Human Cohesive ends or staggered ends Chimeric or recombinant DNA OH P DNA based technologies that enabled the sequencing of whole genomes DNA cloning or recombinant DNA technology Restriction endonucleases DNA sequencing ? genome sequencing 1980 Nobel chemistry 1976 founded Genentech AT tails make cohesive ends. That spawned recombinant DNA technology. WOW! Choice of cloning vectors Copy number of cloned insert Size of cloned insert The constructed E. coli plasmid pBR322 Origin of replication, ~20 copies/cell Two selectable markers, TetR and AmpR Several unique restriction recognition sequences Small size, facilitates entry into cells (transformation). Ideal for cloning small inserts DNA cloning refers to propagating identical copies of a segment of DNA through recombinant DNA technology See fig. 9-4 for positive selection of transformants using drug resistance markers How do you distinguish transformants containing recombinant plasmids from those that religated to form the original vector? Grow on Amp plates Directional cloning using two restriction endonucleases with different cleavage sequences promoter = 48.5 kb Essential genes ~ 30 kb Allows cloning of inserts of up to 23 kb Only recombinant fragments of ~50 kb containing the essential genes and an insert are packaged into viable phage particles Bacteriophage l cloning vector - high yield, ~100 phage/cell Recognition sequences at both ends required for packaging Bacterial artificial chromosomes (BAC) as cloning vectors Insert size 100 kb - 300 kb - one copy per cell Construction of a yeast artificial chromosomes (YAC) Used for cloning DNA segments of up to 2,000 kb. Important for the human genome sequencing project Advantage of YAC over BAC: Bacteria have very efficient recombination systems. Cloned large DNA fragments often undergo rearrangements. How to identify clone of interest from a genomic library? Hybridization or annealing of complementary strands Now, researchers obtain sequence from sequence databases of genes from a range of organisms. Synthesize probe based on known protein sequence
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