PV92 PCR Bio Informatics
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1 Purpose of PCR Chromosome 16 PV92 PV92 PCR Bio Informatics Alu insert, PV92 locus, chromosome 16 Introduce the polymerase chain reaction (PCR) technique Apply PCR to population genetics Directly measure human diversity at the molecular level Compare results to published data online What is PCR? DNA replication gone crazy in a tube! Makes many copies of a specific target sequence from a small amount of template DNA Affects gene mapping, cloning, DNA sequencing and detection Applications in detection of specific mutations, criminal investigations, and human genome What is the needed for PCR? Template (the DNA you want to amplify for the study) Sequence-specific primers flanking the target sequence Forward Reverse Nucleotides Magnesium chloride (enzyme cofactor) Buffer, containing salt Taq polymerase (DNA Polymerase) How does PCR work? 1. Heat (94 o C) to denature DNA strands 2. Cool (60 o C) to anneal primers to template Denaturing Template DNA Heat causes DNA strands to separate Denaturation of DNA at 94 o C 3. Warm (72 o C) to activate Taq polymerase, which extends primers and replicates DNA 4. Repeat multiple cycles 1
2 Annealing Primers Primers bind to the template sequence Taq (DNA) polymerase recognizes doublestranded substrate Primers anneal at 60 o C Taq polymerase extends.. Taq polymerase extends primer DNA is replicated Extend at 72 o C Repeat denaturing, annealing, and extending 40 cycles The exact-length target product is made in the third cycle The target sequence Cycle 1 Cycle 2 PV92 Alu insertion Found on chromosome 16 Cycle 3 View the PCR reaction at the Dolan DNA Learning center: Alu Amplified Region PV92 Alu insertion A member of Alu repeat family Human-specific Alu insertion Intron: Found in a non-coding region of your DNA NOT diagnostic for any disease or disorder Alu Alu repeats Classified as SINEs (Short Interspersed Repetitive Element) Approx. 500,000 Alu copies per haploid genome, representing about 5% of the genome Named for the Alu I restriction site within the element Amplified Region 2
3 Evolutionary Significance of PV92 Alu Inserts Highly conserved Inserted in the last 1,000,000 years Used in population genetics, paternity analysis, and forensics 641 bp PCR Results The PV92 Alu is dimorphic so there are two possible PCR products: No insertion: 641 bp Alu insertion: 941 bp 641 bp 941 bp 300 bp Alu insert Actual Alu PCR Results + - +/- PCR Procedures Day bp 641 bp + - +/- Add Master Mix containing: Nucleotides Primers Reaction buffer Electrophoresis dyes Taq polymerase Day 2 Day 3 Genomic DNA Extraction InstaGene = Chelex cation exchange resin;binds cellular MgCl 2 56 o C loosens connective tissue and inactivates DNases 100 o C ruptures cell membranes and denatures proteins InstaGene Extraction Nuclear membrane Heat disrupts membranes Cell membrane Genomic DNA InstaGene matrix binds released cellular 3
4 Cancer Cancer is a group of diseases caused by loss of cell cycle control. Cancer Cancer is associated with abnormal uncontrolled cell growth. Carcinogens are substances which cause cancer by mutating DNA. Many genes that can mutate to cause cancer control the cell cycle or DNA maintenance (repair). Control of the cell cycle Origin of cancer Cancer begins from the growth of a single abnormal cell. A mutation occurs allowing a cell to undergo cell division when it would not normally divide. Division produces more abnormal cells. Mutations can occur: In somatic cells => sporadic cancer only affecting the individual In germline cells => mutations that are inherited Germline mutations usually require second somatic mutation also. Cell division rates - normal & cancer cells Types of cancer genes Normal cells Bone marrow precursors Lining cells of large intestine Lining cells of rectum Fertilized ovum Hours between cell division Cancer cells Stomach Acute myeloblastic leukemia Chronic myeloid leukemia Lung (bronchus carcinoma) Hour between cell divisions Some cancer cell types grow more slowly than some normal cell types Type of gene Oncogene Tumor suppressor gene DNA repair gene mutation Normal function Promotes division Suppresses cell division Repair DNA mutations Mutated function Promotes division - abnormal time or cell type Fails to suppress division Fail to repair DNA mutations Types of proteins Growth factors Checkpoint molecules Enzymes for mismatch or excision repair 4
5 Characteristics of cancer cells Divide continually (given space and nutrients) Heritable mutations: cells with mutations have daughter cells which inherit the same mutations. Transplantable Dedifferentiated: cells lose their specialized identity Different appearance: reflects dedifferentiation Lack contact inhibition: will divide in a crowd of cells and pile on top of each other Induce angiogenesis (local blood vessel formation) Increased mutation rate Invasive: squeeze into any space available Metastasize: cells move to new location in the body Cancer treatments for breast cancer Strategy Remove cancerous tissue Destroy cancerous tissue Use phenotype to select drug Use genotype to select drug Genomic level Examples Surgery Radiation or chemotherapy to kill dividing cells Estrogen receptor positive women take tamoxifen Her-2/neu positive cancers targeted with herceptin MAb Gene expression profile on DNA microarray to guide drug choice The Human Genome Project Genomics The decade long project to sequence the human genome or determine the order of the nucleotides present in each of the chromosomes end to end. The draft of the human genome was announced in February This represents the work of thousands of researchers in an international collaboration. Human Genome Project: uses of genetic information The Human Genome Project Before sequencing the human genome, a framework map of the genome was needed. Correspondences between cytogenetic information, genetic linkage information, and physical information was determined. 5
6 Two methods for sequencing the human genome Sequencing the human genome Isolated DNA was broken into smaller pieces and cloned into vectors. In the public genome project, restriction digests of cloned fragments were compared to determine the order of the pieces along the chromosomes. Subfragments were sequenced and assembled into one continuous sequence. In the corporate genome project, fragments were randomly sequenced and the sequences were aligned and assembled into one continuous sequence afterwards. (This effort also used information established in the public project.) Steps in genome sequencing and analysis Obtain chromosome maps with landmarks from linkage or cytogenetic studies or RFLP sites Obtain chromosome pieces in gene libraries or shotgun entire sequence Sequence the pieces Overlap aligned sequences Compare sequence to other species 6
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