Chapters 20 and 21 Notes
Chapters 20 and 21 Notes BIOL 2601 - 01
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BIOL 2601 - 01
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This 6 page Class Notes was uploaded by Suzanne Notetaker on Wednesday November 11, 2015. The Class Notes belongs to BIOL 2601 - 01 at Youngstown State University taught by Dr. Asch in Fall 2015. Since its upload, it has received 21 views. For similar materials see General Biology in Biology at Youngstown State University.
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Date Created: 11/11/15
Chapter 20 Genetic Technology Gene Cloning procedures that lead to many copies of a particular gene Recombinant DNA technology use of lab techniques to isolate and manipulate fragments of DNA Recombinant DNA DNA from two or more sources Once inside host cell recombinant molecules replicated to produced identical copies or clones Why 9 for study or manipulation and obtain large amounts of gene product Step 1 isolate vector DNA from bacterial cell and gene of interest from chromosomal DNA Vector DNA is a carrier for DNA segment to be cloned When vector introduced into living cell it can replicate Common vectors are plasmid or viral Step 2 insert chromosomal DNA into vector Cut DNA using restriction enzymes cut made by bacteria as protection against bacteriophages Cut at specific known restriction sites Most restriction sites are palindromic May produce sticky ends DNA ligase use to permanently link DNA and paste Want recombinant vector with gene inserted Step 3 goal to have recombinant vector taken up by bacteria bacteria will replicate vector and divides to produce many cells Vector carries a selectable marker Adding antibiotics selects for cell expressing antibiotics resistance gene ampR AmpR codes for betalactamase that degrades ampicillin normally kills bacteria Growth on ampicillin plates indicates bacteria contain plasmid with the selectable marker Eliminate recircularized empty vectors lacZ gene is built into vector Insertion of cloned DNA disrupts lazZ gene LacZ codes for betagalactosidase which cleaves colorless XGal into blue dye Recirularized plasmids form blue colonies Desired recombinant vectors from white colonies DNA Library collection of many recombinant vectors each with a fragment of chromosomal DNA Types 1 Genomic library inserts derived from chromosomal DNA 2 CDNA library use reverse transcriptase to make complementary DNA CDNA from mRNA lacks introns simpler to use Electrophoresis separate macromolecules on a gel separate DNA or proteins based on their charge sizelength and mass Polymerase chain reaction PCR9amplifies DNA make many copies Ingredients 1 Primers match sequences at each end of fragment 2 dNTPs deoxynucleotide triphosphates 3 Taq polymerase heatstable from of DNA polymerase DNA runs through repeated cycles of denaturation annealing and synthesis by thermocycler machine Denaturation heat DNA to separate strands Primer annealing lower temp to allow primers to bind to template DNA Primer extension incubate at higher temp synthesis of complementary strand After 30 cycles DNA sample Will increase 230fold Genomics molecular analysis of entire genome of a species Phases 1 Mapping of genome determine relative locations of inserted chromosomal pieces Bacterial artificial chromosomes BAC9contain up to 500000 base pairs Yeast artificial chromosomes YAC9up to 2 million base pairs 2 Functional genomics how DNA impact structure and function expression analysis Contig contiguous9series of clones that contain overlapping pieces of chromosomal DNA overlap helps us identify order of fragments along chromosomes DNA sequencing determines base sequencing of DNA Dideoxy chaintermination method Dideoxynucleoside triphosphates ddNTPs missing 3 OH group chain terminate 4 tubes With copies of single stranded DNA different labeled nucleotide DNA polymerase make complementary strand until ddNTP inserted and chain terminates After electrophoresis DNA sequence read by determining the base at end of strand Biotechnology use of living organisms to benefit humans Insulin 19829human insulin by recombinant bacteria Prior to 19829isolated by cattle Composed of 2 polypeptides A and B Coding sequence inserted into E coli Fusion proteins extracted and Betagalactosidase removed Purified A and B chain mixed to form functional protein Transgenics organism carries genes using molecular technology Genetically Modified Organisms GMOs Gene replacement cloned gene recombines With normal gene on a chromosome 1 or 2 copies replaced creating hetrozygote Heterozygotes crossed to yield homozygotes Gene knockout if cloned gene mutated and inactivates function homozygote Will not have gene function Very useful for studying human disease Cloning in mammals Plants cloned from somatic cells Dolly first cloned lamb 1996 Mammary cells from adult sheep removed Fused diploid mammary cell With enucleated sheep oocyte unfertilized Zygote implanted Dolly and donor almost genetically identical different in mitrochondrial DNA and maternal effect genes Cloning now achieved in several mammal species DNA Fingerprinting identifies and distinguishes among individuals based on variations in their DNA Chromosomes DNA produces series of unique pattern of bands on a gel Automated using PCR to amplify short tandem repeat sequences STRs STRs found at specific locations in genome Uses identify species of bacteria and fungi forensics paternity testing Chapter 21 Genomes Proteomes and Bioinformatics Bacterial and Archaeal Genomes Evolution unifying theme of biology Prokarvotic Genomes Important because Bacteria cause disease apply knowledge to more complex organisms and first eukaryotic cell involved union between archaeal and bacterial cell Entire genomes have been sequenced and analyzed Less complex than eukaryotes Lack centromeres and telomeres Single origin of replication Little repetitive DNA Chromosomes usually several hundred thousand to few million base pairs Most contain single chromosome multiple copies in cell some with different chromosomes Chromosomes usually circular some linear some have both Often have plasmids typically small Venter Smith and Colleagues Sequenced the First Complete Genome Haemophilus in uenza Causes a variety of human diseases One strategy for mapping large genome extensive mapping Alternative Shotgun DNA sequencing Randomly sequence fragments No extensive mapping but waste time sequencing same DNA region Eukarvotic Genomes Found in sets of linear chromosomes Extranuclear DNA found in mitochondria and chloroplasts Sequenced for many species Four Motivators to Sequence Genomes 1 Identify and characterize genes in model organisms 2 More info to identify and treat human diseases 3 Improve strains of agricultural species 4 Establish evolutionary relationships Genome size is NOT the same as number of genes Increase in amount of DNA correlated with increasing cell size cell and body complexity Repetitive Sequences Eukaryotic genomes many copies of short DNA sequences 1 Moderately repetitive few hundred to several thousand times a rRNa genes multiple origins of replication or role in gene transcription and translation 2 highly repetitive tens of thousands or millions of times a most no known functions Noncoding vs Coding DNA 98 of genome is noncoding Intron unique noncoding and repetitive DNAs 2 of genome is coding Exons of structural genes and genes for rRNA and tRNA Transposable Elements Transposition short segment of DNA moves from original site to new site Transposable elements TEs DNA that move jumping genes found in all species examined First discovered by Barbara McClintock 1 DNA transposons both ends have inverted repeats DNA sequences identical but run in opposite directions a Transposase enzyme that facilitates transposition b CutandPaste mechanism i Transposase recognizes inverted repeat then remove sequence from original site ii Complex moves to new site where transposase inserts it into chromosome 2 RNA intermediates retroelements or retrotransposons in eukaryotes a Contains reverse transcriptase and transposase i Transcriptase uses RNA as template to make copy of DNA b May accumulate rapidly in genome Alu are 10 of human genome Role of TEs 1 Exist because they have characteristics that allow them to insert themselves and replicate can do harm Q 2 May benefit a species new genetic combinations Human Genome Proiect Oct 1 1990 to end of 2003 Goals Identify all human genes Sequence entire human genome Develop technology Analyze genomes of model organisms Develop legal ethical and social programs addressing results U PP Pi Proteomes collection of proteins that a given cellspecies makes Protein abundance can refer to Number of genes for type of protein Amount of each protein made Ex Liver cell vs Muscle cell Same genes abundance very different Proteomes are Larger than Genomes due to 1 Alternative splicing single premRNA spliced into more than one version 2 Posttranslational covalent modification a Permanent or reversible b Involved in assembly and construction of proteins 0 Phosphorylation methylation acetylation reversible Bioinformatics use computers and math tools to record store and analyze biological information Highly interdisciplinary 1St Step collect and store data 2nd Step write programs to analyze sequences in particular ways Translate DNA sequence into amino acid sequence for all 3 reading frames Look at results for both strands Databases large number of files and store them in one place for rapid search and retrieval Nucleotide sequences 1 GenB ank US 2 EMBL Europe 3 DDBJ Japan Amino acid sequences SwissProt Swiss protein PIR Protein TrEMBL translated sequences from EMBL Genpept translated sequences from GenBank P9P Identify Homologous Sequences Software can identify evolutionarily related genes Closely related organisms tend to have genes with similar DNA sequences Ortholog homologous genes in different species Reveal evolutionary relationships BLAST Basic Local Alignment Search Tool9uses particular genetic sequence to find homologous sequences in large database
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