* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download cDNA libraries, Microarray Analysis
Zinc finger nuclease wikipedia , lookup
Genetic engineering wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Epitranscriptome wikipedia , lookup
DNA profiling wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Genome evolution wikipedia , lookup
Non-coding RNA wikipedia , lookup
Metagenomics wikipedia , lookup
Gene expression profiling wikipedia , lookup
Human genome wikipedia , lookup
Epigenetics of human development wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Cancer epigenetics wikipedia , lookup
History of RNA biology wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Point mutation wikipedia , lookup
Nutriepigenomics wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
SNP genotyping wikipedia , lookup
DNA polymerase wikipedia , lookup
DNA vaccination wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Designer baby wikipedia , lookup
Genomic library wikipedia , lookup
Microsatellite wikipedia , lookup
Epigenomics wikipedia , lookup
DNA supercoil wikipedia , lookup
Microevolution wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Molecular cloning wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Non-coding DNA wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
History of genetic engineering wikipedia , lookup
Primary transcript wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Helitron (biology) wikipedia , lookup
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? Eukaryotic genes have introns that interrupt the protein coding sequence. 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? 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 Tissue sample Isolate mRNA. Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope slide on which copies of singlestranded 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. 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 5 3 Target sequence Genomic DNA Denaturation: Heat briefly to separate DNA strands 3 5 5 3 Bookmark 3 Cycle 1 yields 2 molecules 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 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence 5 Primers New nucleotides Gene Therapy Bookmark