Biotechniques.docx - Technique What it does Benefits...

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Technique What it does Benefits/ detections Requirements Misc Examples Restriction Enzymes Cuts at palindromes to produce sticky or blunt ends Digests DNA into a characteristic and reproducible collection of fragements Need DNA ligase to connect ends, enzyme that is going to cut the DNA EcoRI cuts the human genome Vector DNA molecule that replicates autonomously in a bacteria or yeast host for subsequent purification Allow for partitioning of genome into manageable fragments for sequencing Have to have a bacteria to put the vector in, Plasmids – for small fragments BACs – designed for larger fragments Expression vectors -- Used to express full- length cDNAs in bacteria, yeast or cultured cells – contain a promoter and other signals for efficient transcription and translation Southern Blotting Standard method for analyzing particular DNA fragments cleaved by restriction enzymes Can detect large deletions or insertions Need DNA, gel electrophorsis, Denature DNA Smallest fragments travel faster Detection of a deletion of the X-linked androgen receptor Allele-Specific Oligonucleotide Synthesized to match either the normal DNA sequence or the mutant gene sequence hybridizes only to one or the other Can detect single nucleotide changes which cannot be distinguished by conventional Southern Blot Analysis Have to know the specific sequence of the mutation , ASO probe specific to the sequence A failure to hybridize to a specific mutant

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