MBIO 3410 lecture Nov 8 2007

MBIO 3410 lecture Nov 8 2007 - Last Lecture Overview PCR...

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1 Last Lecture Overview PCR PCR Primer Mismatches PCR- based Mutagenesis PCR to search for homologous genes Degenerate Primers PCR in the Diagnosis of Genetic Disease PCR to identify insertion/ deletion mutations Cloning PCR Products RT–PCR Relative quantitation Absolute quantitation Real-time PCR TaqMan® Real-time PCR Quantification Applications of PCR DNA Libraries Genomic DNA library cDNA libraries
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2 Genomic Libraries
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3 Genomic Libraries Chromosomes contain an enormous quantity of DNA, too large to be cloned efficiently into any of the vectors currently available It is necessary to fragment the DNA before it is cloned into a vector DNA should be broken up into random and overlapping fragments prior to cloning Such cleavage would ensure that the library contains representative copies of all DNA fragments present within the genome There are two basic mechanisms for cleaving DNA that are used in the construction of genomic libraries Mechanical shearing Restriction enzyme digestion
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4 Mechanical shearing Purified genomic DNA is either passed several times through an narrow-gauge syringe needle or subjected to sonication to break up the DNA into suitable size fragments that can be cloned Typically, an average DNA fragment size of about 20 kb is desirable for cloning into λ based vectors Mechanical methods such as these have the advantage that DNA fragmentation is random, but suffer from the fact that large quantities of DNA are required, and that the average DNA fragmentation size may be quite variable The ends produced are likely to be blunt ends due to the breakage across both DNA strands which can be repaired with Klenow polymerase If the ends are not blunt, DNA polymerase will fill in any recessed 3’-ends on DNA molecules if dNTPs are provided
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5 Restriction enzyme digestion DNA libraries are usually constructed by digesting the genomic DNA with restriction enzymes via partial restriction digests This ensure that not all DNA recognition sequences are cut In practice, partial restriction digestion is normally performed using a restriction enzyme that recognize and cleave very commonly occurring sequences
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6 Sticky-ended DNA is considerably more efficient than blunt ends so it is desirable to generate genomic fragments that contain sticky ends in the cloning process This can be achieved two ways: 1. Linkers or adaptors 2. Restriction enzymes that generate sticky ends Blunt ended DNA fragments can be ligated to: oligonucleotides that contain the recognition sequence for a restriction enzyme = linkers or possess one blunt end for ligation to the genomic DNA and an overhanging sticky end for cloning into particular restriction sites = adaptors Choice of Restriction Enzyme
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7 DNA fragments are first protected from restriction enzyme cleavage by treatment with EcoR I methylase, in the presence of S- adenosylmethoinine
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