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Molecular biology techniques

Molecular biology techniques - 1 Aim SourceofDNA Steps...

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1. PCR from genomic DNA/cDNA Aim: Detection of specific sequence (or set of specific sequences) Source of DNA: Genomic + cDNA Steps: > amplification by PCR a) Quantitative > measure/compare amounts of PCR product b) Qualitative > sequence PCR product (“send for sequencing” Examples of applications: a) Comparison of levels of transcript between different cell types Detection of increased transcription of oncogene in tumor cell Study of changes in gene expression during embryonic development b) Characterization of DNA sequence in mouse/Drosophila mutant Detection of human genetic diseases Study of alleles of a particular gene in a animal/human/plant population Systematics: comparison of various species using sequences from a set of genes (e.g. mitochondrial genes for short term evolution) DNA fingerprint Analysis of spliced variants (which of the possible isoforms is expressed,…) 2. Libraries Aim: Creating a collection of DNA fragments Source of DNA: Genomic + cDNA Steps: > ligation of DNA fragments in vector (plasmid for small pieces, BACs or cosmids for large pieces) Bacteria transformation Isolation of clones Examples of applications: Screen to isolate cDNA sequences Large scale sequencing (in this case, random picking of many clones, growth of bacteria to obtain enough DNA, and sequencing > Databases
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