From Haystacks to Needles AP Biology Fall 2010

Isolating Genes  Gene library: a collection of bacteria that house different cloned DNA fragments, one of which is of interest May be the entire genome or of cDNA, which is free of introns

Gene Library

Isolating Genes  A particular gene of interest can be isolated from other genes by use of probes

Isolating Genes  DNA probes: short DNA sequences assembled from radioactive nucleotides Can pair with parts of the gene to be studied  This nucleic acid hybridization technique can be used with other procedures to select cells and their DNA May be of interest to a researcher

Isolating Genes  If researchers know the desired gene sequence, they use it to construct a primer by synthesizing the single stranded DNA  If the sequence is unknown, they use DNA from the same gene of a closley related species that has already been isolated

Big Time Amplification – PCR  The polymerase chain reaction (PCR) can be used to make millions of copies of cDNA

Big Time Amplification – PCR  What is needed for the reaction? Primers are short nucleotide sequences that are made in the laboratory They are recognized by DNA polymerases as the START tags for building complementary sequences of DNA dictated by computer programs stored in the machines

Big Time Amplification – PCR  What are the reaction steps? 1. Researchers mix primers, DNA polymerase, cellular DNA from an organism, and free nucleotides

Big Time Amplification – PCR 2. Precise temperature cycles cause the DNA strands to separate, exposing the bases

Big Time Amplification – PCR 3. Primers become positioned on the exposed nucleotides to form new copies of the original DNA

Big Time Amplification – PCR 4. Each round of reactions doubles the number of DNA molecules to eventually produce billions of molecules from very tiny amounts of original DNA