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Lecture 9

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BIOL 205

Recombinant DNA What is needed? • A tool to cut DNA into pieces (enzymes or mechanical breakage) • A means of identifying and purifying DNA pieces of interest! • Enzymes : to make and manipulate DNA • A means to replicate DNA (so we have lots of DNA to work with). What types of DNA can we work with ? 1. GENOMIC DNA – DNA extracted from chromosomes -needs to be cut up before use! 2. cDNA (Complementary DNA) 5’ AAAAA 100-200 mRNA (isolated from cytoplasm) TTTTT Reverse Anneal an oligo-dT Transcriptase 18 RNA/DNA hybrid Makes a DNA copy DNA polymerase TTTTT Cleave the End loop of Double-stranded DNA=cDNA ssDNA 3. Chemically synthesized DNA What does a gene look like in a eukaryote/mammalian genome ENZYMES USED IN MOLECULAR GENETICS 1. RESTRICTION ENZYMES- cut DNA into defined segments 2. DNA LIGASE – join fragments of DNA 3. DNA POLYMERASES- synthesize DNA on a template 4. REVERSE TRANSCRIPTASE – copy RNA into DNA 5. NUCLEASES – deoxyribonucleases and ribonucleases exonuclease –cleaves from the ends 5’or 3’ endonuclease-cleaves internally 6. RNA POLYMERASE- make RNA on a template 7. TERMINAL TRANSFERASE – add a nucleotide 8. POLYNUCLEOTIDE KINASE –add a phosphate 9. ALKALINE PHOSPHATE - remove a phosphate RESTRICTION ENZYMES Cleave DNA in a sequence-specific manner! Evolved as a defense mechanism---protect against the invasion of foreign DNA Different bacteria make different restriction enzymes Nomenclature: Eco RI E co R I Species Strain Enzyme # Genus coli Escherichia Many restrictionsites recognize palindromicsequences Palindromes: Same sequence read forward as backward In English : kayak, rotator, radar Able was I ere I saw Elba Eco RI -6 bp cleavage recognition site 5’ GAATTC 5’ G-OH 5’ AATTC CTTAAG 3’ CTTAA- 5’ G 4 bp ssDNA overhang If sequences were random the distributionfrequency would be: 4 base cutter = 4 = 1 cut every 256 bp 6 base cutter= 4 = 1 cut every 4096 bp 8 8 base cutter= 4 = 1 cut every 65,536 Cut DNA can be separated by agarose or polyacrylamide gel electrophoresis SYBR GREEN STAINING SAFER Ethidium Bromide stained gel Fluorescent under UV light Ethidium bromide intercalates between DNA strands Formation of a recombinant DNA molecule Figure 20-2 Vector Features • Must contain a replicon that enables it to replicate in host cells (region of DNA that is amplified, i.e.: has origin of replication) • Several marker genes • Unique cleavage site(s) • For expression, must contain control elements, such as promoters, terminators, ribosome binding sites, etc… Cloning Vectors Vector Max. Insert Size(kb) Plasmid ~5 Cosmid ~45 Lambda ~20 (text says 50) P1 phage ~100 BAC ~350 YAC ~1000 BAC/Y AC
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