* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download What does PCR stand for?
Nutriepigenomics wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
DNA paternity testing wikipedia , lookup
DNA barcoding wikipedia , lookup
Oncogenomics wikipedia , lookup
Genetic engineering wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Human genome wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
DNA profiling wikipedia , lookup
DNA polymerase wikipedia , lookup
Metagenomics wikipedia , lookup
Point mutation wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
DNA vaccination wikipedia , lookup
Designer baby wikipedia , lookup
Primary transcript wikipedia , lookup
Genomic library wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Non-coding DNA wikipedia , lookup
Microevolution wikipedia , lookup
DNA supercoil wikipedia , lookup
Epigenomics wikipedia , lookup
Molecular cloning wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genome editing wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Helitron (biology) wikipedia , lookup
History of genetic engineering wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
SNP genotyping wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Microsatellite wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Polymerase Chain Reaction a.k.a. “How’d they get all that DNA from just a little blood?” What does PCR stand for? Polymerase Chain Reaction Developed by Kary Mullis – Nobel Prize Received a $20,000 bonus; later sold it to Hoffman-LaRoche for $300,000,000. What is the goal of PCR? To make many copies of a small section of DNA. DNA Synthesis in vitro (in a test tube). How does PCR work? Very Similar to DNA Synthesis How does PCR work? http://www.lsic.ucla.edu/ls3/tutorials/gene_clonin g.html http://www.iupui.edu/~wellsctr/MMIA/htm/animati ons.htm DNALC http://wps.prenhall.com/wps/media/objects/487/4 98929/CDA12_2/CDA12_2a/CDA12_2a.htm What is needed for a PCR reaction? DNA template – DNA to be copied; “target sequence” What is needed for a PCR reaction? PCR primers – short DNA sequences that bind to DNA; are complimentary to beginning and end of target sequence What is needed for a PCR reaction? Taq Polymerase Polymerase copies DNA From heat loving bacteria Thermus aquaticus. Can survive hot temperatures needed in PCR reaction. Always copies in 5’ 3’ direction What is needed for a PCR reaction? Nucleotides – building blocks of DNA Thermalcycler – computerized to change temperatures What are the 3 main steps of PCR? Denaturing – DNA strands separate at hydrogen bonds Temperature = 950C What are the 3 main steps of PCR? Annealing – Primers bind Temperature is dependent on primer sequence Annealing temperature = 3(G+C) + 2(A+T) # of H-bonds between nucleotides What are the 3 main steps of PCR? Extension – DNA is copied Temperature = 720C PCR Reaction Summary 1. Denaturing 2. Annealing 3. Extension 30 cycles How many times can target DNA be copied? DNALC What would someone do with PCR? Forensics Identification of corpses or body parts. Analysis of pathogens. Eliminate or link suspects to scene of crime through a sample (blood, human hair, skin, semen) left at scene or circumstantial links (pet hair, plant parts). What would someone do with PCR? Medical (genotyping) Genetic diseases identified pre- or post-natally. Cancers identified and classified. Identifying bacterial or viral strains for proper treatment. What would someone do with PCR? Food Science Samples can be tested for genetic engineering traits. Bacterial contaminants and source of contamination can be quickly identified. Pedigrees and traits of valuable animal food stocks can be verified. What would someone do with PCR? Relationships Paternity. Evolution – developing relationships between organisms or studying extinct species from museum specimens. Ecology – tracking organisms or defining biodiversity by identifying unique species. PCR Review Music Video BioRad “When You Need to Find Out Who The Daddy Is” BioRad “GTCA” PCR Lab #1 - Alu What DNA sequence are we going to copy? Alu element Chromosome 16 Does NOT code for a gene PCR Lab #1 - Alu 95% of the human genome does NOT code for proteins “Junk” DNA Nobody knows why we have it All of it is copied during DNA synthesis and passed from one generation to the next PCR Lab #1 - Alu Alu is an example of a “jumping gene” (also known as a transposon) Come from a virus infection All primates share the same initial Alu sequence Human chromosomes contain an estimated 1,000,000 Alu copies equaling 10% of the total genome. PCR Lab #1 - Alu Alu elements are dimorphic (meaning two forms) Chromosome 16 either has this Alu element or it doesn’t Since chromosomes come in pairs, a person is either ++, +-, or -- . DNA sequences such as Alu are used in diagnosis of genetic disease, forensic identification, and paternity testing. PCR Lab #1 – PTC Taster What DNA sequence are we going to copy? PTC taste receptor gene (produces a bitter taste) PCR Lab #1 – PTC Taster Chance discovery PCR Lab #1 – PTC Taster Being a “taster” is a dominant trait Homozygous dominant – TT TASTERS Heterozygous – Tt Homozygous recessive - tt NON-TASTERS PCR Lab #1 – PTC Taster About 70% of people are tasters 58% for Aboriginal people of Australia 98% of Native Americans PCR Lab #1 – PTC Taster Non-taster PCR product - 221 bp Taster PCR product - 221 bp SNP – Single Nucleotide Polymorphism. One “letter” makes a big difference. PCR Lab #1 – PTC Taster What will be our DNA source? Cheek cells Rinse our mouths with a saline solution (0.8% NaCl) PCR Lab #1 – PTC Taster Why not just use pure water to rinse our mouths? What would happen to our cells in 100% water?) Due to osmosis, cells would burst before we could collect DNA PCR Lab #1 – PTC Taster Cells are collected, allowed to settle, and then mixed with Chelex solution. Negatively charged beads – removes Mg2+ which inhibits Taq polymerase. PCR Lab #1 – PTC Taster Chelex, cheek cell mixture is heated to 980C. Heat bursts cells open and cell debris is bound to Chelex beads. Chelex beads and cell debris is heavy so it will settle to bottom of tube. We will collect the supernatant (contains DNA) and will freeze it. Set up PCR reaction on Thursday. 3’ A T C G G A C C G A C T G C A T G C C A G T T A A T A G C C A A T 5’ 5’ T A G C C T G G C T G A C G T A C G G T C A A T T A T C G G T T A 3’ Denature; 950C 3’ A T C G G A C C G A C T G C A T G C C A G T T A A T A G C C A A T 5’ Taq Pol Anneal; Extend; ~60 7200CC 5’ C T G G 3’ 3’ T A G C 5’ Taq Pol 5’ T A G C C T G G C T G A C G T A C G G T C A A T T A T C G G T T A 3’ PCR Lab #2 - mtDNA What DNA sequence are we going to copy? mtDNA control element Does NOT code for a gene PCR Lab #2 - mtDNA In which organelle is this DNA stored? mitochondria All DNA is NOT stored in nucleus! PCR Lab #2 - mtDNA How many genes are on this DNA? 37 genes in 16,569bp PCR Lab #2 - mtDNA What proteins do these genes make? Proteins involved in cellular respiration PCR Lab #2 - mtDNA Interesting facts regarding mtDNA Always inherited from mom Mutates at a constant rate PCR Lab #2 - mtDNA PCR Lab #2 - mtDNA Compare sequences to determine genetic relationships (phylogenetic tree) PCR Lab #2 - mtDNA Interesting facts regarding mtDNA Mitochondria may have been a bacterial cell at some point