Additional Powerful Molecular Techniques Synthesis of cDNA (complimentary DNA) Polymerase Chain Reaction (PCR) Microarray analysis Link to Gene Therapy.
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Additional Powerful Molecular Techniques Synthesis of cDNA (complimentary DNA) Polymerase Chain Reaction (PCR) Microarray analysis Link to Gene Therapy information (in syllabus)
cDNA: a DNA compliment of RNA template Why: DNA is more stable than RNA and DNA copies of RNA can be cloned into vectors -First strand synthesis uses RNA as a template to make a complimentary single stranded DNA -Second strand synthesis uses first DNA strand as a template to make double stranded cDNA for cloning Bookmark
What is the source of the RNA? Often mRNA What is a major difference between prokaryotic & eukaryotic genes? If you were interested in eukaryotic protein coding sequences, why would it be advantageous to use cDNA clones or libraries rather than genomic clones or libraries? Eukaryotic genes have introns that interrupt the protein coding sequence. Since cDNA is made from mature mRNA, no introns!
Studying Expression of Interacting Groups of Genes Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions
LE 20-14 Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope slide on which copies of single- stranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. Isolate mRNA. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA microarray Size of an actual DNA microarray with all the genes of yeast (6,400 spots)
Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules
LE 20-7 Genomic DNA Target sequence 5 3 3 5 5 3 3 5 Primers Denaturation: Heat briefly to separate DNA strands Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Extension: DNA polymerase adds nucleotides to the 3 end of each primer Cycle 1 yields 2 molecules New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Bookmark