Chapter 20: Biotechnology. 1. DNA Sequencing 3/26/2017. DNA Sequencing. 2. DNA Cloning. 3. Studying Gene Expression. 4. Manipulating Genomes

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1 hapter 0: Biotechnology 1. DN Sequencing. DN loning 3. Studying ene Expression 4. Manipulating enomes 5. herapeutic & Diagnostic echniques 1. DN Sequencing hapter Reading pp EHNIQUE DN (template strand) DN Sequencing Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) dp ddp dp ddp DN polymerase dp dp P P P P P P OH ddp ddp H DN replication in vitro using 1 of 4 different chainterminating dideoxynucleotides (ddnps) results in a set of DN fragments ending in all positions with,,, or that can be resolve by length on a gel 1

2 DN Sequencing uses hain erminators Normal nucleotide (dnp) Dideoxynucleotide (ddnp) * DN synthesis is carried out in reactions containing the following: DN template to be sequenced dnp s DN primer DN polymerase ddp, ddp, ddp or ddp each ddnp is labeled with a different fluorescent tag he color reveals the identity of the ddnp that terminated DN synthesis at each position, thus revealing the sequence! EHNIQUE (continued) DN Sequencing DN (template strand) Direction of movement of strands Labeled strands dd dd dd dd dd dd Shortest Longest labeled strand Detector dd dd dd Longest Laser Shortest labeled strand

3 DN Sequencing Direction of movement of strands Longest labeled strand Detector RESULS Laser Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand Shortest labeled strand Next eneration Sequencing. DN loning hapter Reading pp

4 What is Recombinant DN? he joining of DN from different sources. his can happen in nature (in vivo) the transfer of DN involving bacteria or viruses or in the laboratory (in vitro) the cutting & splicing of DN fragments by molecular biologists he term recombinant DN generally refers to the in vitro kind which is commonly called gene cloning Bacterium ene loning 1 ene inserted into plasmid ell containing gene of interest Bacterial chromosome Plasmid Recombinant DN (plasmid) Recombinant bacterium ene of interest Plasmid put into bacterial cell DN of chromosome ( foreign DN) gene of interest is inserted into a plasmid vector bacterial host transformed with recombinant plasmid bacterial clone with recombinant plasmid is identified loned gene can then be used in a variety of ways. ene of interest opies of gene Basic research on gene ene loning 4 Basic research and various applications 3 Host cell grown in culture to form a clone of cells containing the cloned gene of interest Protein expressed from gene of interest Protein harvested Basic research on protein ene for pest ene used to alter resistance inserted bacteria for cleaning into plants up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth 4

5 DN fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction site DN 1 Restriction enzyme cuts sugar-phosphate backbones. Sticky end 3 DN ligase One possible combination seals strands Recombinant DN molecule Restriction Enzymes ene cloning usually involves the use of restriction enzymes that cut DN at very specific sequences: e.g. EcoRI cuts at: here are many different restriction enzymes, each cutting a different sequence he ene loning echnique EHNIQUE Bacterial plasmid amp R gene cut plasmid & DN to be cloned with same RE ligate vector & DN insert with DN ligase lacz gene Restriction site Sticky ends Recombinant plasmids Hummingbird cell ene of interest Hummingbird DN fragments Nonrecombinant plasmid transform bacteria with ligated DN Bacteria carrying plasmids Selection for Bacterial lones ntibiotic resistance allows selection for bacterial clones containing the plasmid vector. RESULS olony carrying nonrecombinant plasmid with intact lacz gene Bacteria carrying plasmids olony carrying recombinant plasmid with disrupted lacz gene One of many bacterial clones Blue/white selection via lacz gene disruption allows identification of clones containing a DN insert. 5

6 EHNIQUE 1 Mixture of DN molecules of different sizes Longer molecules RESULS Power source athode node Wells Power source el Shorter molecules el Electrophoresis Separation of DN fragments through a porous gel matrix: gel is either agarose or polyacrylamide electric current pulls negatively charged DN toward the positive pole rate of movement is inversely proportional to DN fragment size DN Libraries collection of cloned genes from an organism is called a DN library. enomic DN Library a collection of chromosomal DN fragments cloned into a particular vector essentially cloned pieces of the organism s genome cdn Library a collection of DN fragments produced from messenger RN (mrn) cloned into a vector produced from mrn by reverse transcriptase a collection of expressed genes with NO intron DN DN Libraries Foreign genome ut with restriction enzymes into either small fragments or large fragments Bacterial artificial chromosome (B) Large insert with many genes Recombinant plasmids (b) B clone Plasmid clone (a) Plasmid library (c) Storing genome libraries 6

7 DN in nucleus mrns in cytoplasm Reverse transcriptase mrn Poly- tail DN Primer strand DN polymerase cdn Producing cdn cdn (DN complementary to mrn) is produced as follows: purify mrn fr. desired cell type treat with reverse transcriptase and oligo-d primer results in double-stranded DN copies of all mrn from the cell clone cdn fragments into vector such as a plasmid transform bacterial hosts Results in a collection of cloned cdn corresponding to coding sequences of all expressed proteins EHNIQUE Multiwell plates holding library clones Screening DN Libraries Radioactively labeled probe molecules ene of interest Probe DN Singlestranded DN from cell Film Nylon membrane Location of DN with the complementary sequence Nylon membrane DN from library clones immobilized on a membrane radioactively labeled DN similar in sequence to desired clone is added as a probe hybridization of probe to complementary DN IDs clone PR he Polymerase hain Reaction is a technique to selectively amplify a desired DN sequence: essentially DN replication in vitro results in exponential amplification of target DN sequence artificial primers specific for target DN sequence limit replication to DN complementary to primers EHNIQUE ycle 1 yields molecules ycle yields 4 molecules ycle 3 yields 8 molecules; molecules (in white boxes) match target sequence enomic DN 1 Denaturation nnealing 3 Extension arget sequence Primers New nucleotides 7

8 Overview of PR Every PR reaction requires the following: 1) source of target DN template ) artificial primers flanking DN of interest 3) heat-stable DN polymerase (from hyperthermophile) 4) dnp s 5) automated thermocycler to facilitate repeated: denaturation of DN (separating the strands) hybridization of primers to template DN synthesis Resulting PR products can then be analyzed by gel electrophoresis. PR loning involves designing restriction sites into the primers to facilitate cloning 3. Studying ene Expression hapter Reading pp ,

9 EHNIQUE 1 cdn synthesis PR amplification 3 el electrophoresis RESULS Primers mrns -globin gene cdns Embryonic stages R-PR produce cdn with reverse transcriptase carry out PR using specific primers for desired gene gel electrophoresis of resulting PR fragments mount of DN reflects amount of original mrn sequence. in situ Hybridization in situ Hybridization How is in situ hybridization carried out? fluorescently labeled anti-sense RN probes added to tissue sample hybridization with complementary mrn sequences location of specific gene expression within the tissue (i.e., in situ) is revealed 9

10 DN Microarrays DN microarray is a solid surface containing a precise array of single-stranded DN sequences from 1000s of different genes in an organism. labeled cdn is produced from test cells and allowed to hybridize with sequences in the array intensity of signals reveal expression of specific genes within the test cells. When cdn from different sources is labeled differently, gene expression from each source can be compared in a single microarray (as shown on the slide after next). EHNIQUE 1 Isolate mrn. issue sample Make cdn by reverse transcription, using fluorescently labeled nucleotides. mrn molecules nalyzing gene expression using a DN microarray. 3 pply the cdn mixture to a microarray, a different gene in each spot. he cdn hybridizes with any complementary DN on the microarray. Labeled cdn molecules (single strands) DN microarray DN fragments representing a specific gene 4 Rinse off excess cdn; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. DN microarray with,400 human genes omparing ene Expression 10

11 Single Nucleotide Polymorphisms (SNPs) SNPs are single nucleotide differences that correspond with specific disease-causing alleles. PR and hybridization techniques (e.g., microarrays) can reveal the presence of such alleles (genetic testing). DN SNP Normal allele Disease-causing allele 4. Manipulating enomes hapter Reading pp. 4-45, loning Plants Some plants can be cloned from a single adult plant cell. ross section of carrot root -mg fragments Fragments were cultured in nutrient medium; stirring caused single cells to shear off into the liquid. Single cells free in suspension began to divide. Embryonic plant developed from a cultured single cell. Plantlet was cultured on agar medium. Later it was planted in soil. dult plant 11

12 EXPERIMEN Frog embryo Frog egg cell Frog tadpole his experiment produced the first artificially cloned animal. Less differentiated cell Donor nucleus transplanted UV Enucleated egg cell Egg with donor nucleus activated to begin development Fully differentiated (intestinal) cell Donor nucleus transplanted RESULS loning nimals Most develop into tadpoles. Most stop developing before tadpole stage. EHNIQUE Mammary cell donor 1 4 rown in culture 5 6 RESULS ultured mammary cells Implanted in uterus of a third sheep Embryonic development Egg cell from ovary 3 ells fused Egg cell donor Nucleus removed Nucleus from mammary cell Early embryo Surrogate mother Lamb ( Dolly ) genetically identical to mammary cell donor he first cloned mammal, Dolly the sheep, was cloned by this method. Other mammals such as cats have since been cloned and presumably humans could be cloned this way as well. ransgenic Organisms ransgenic organisms have a foreign gene (e.g. from another species) inserted into their genome. also known as genetically modified organisms (MOs) 1

13 RISPR-as9 a new method to modify genomes as9 active sites uide RN complementary sequence NULEUS as9 protein uide RN engineered to guide the as9 protein to a target gene Part of the target gene omplementary sequence that can ctive sites that bind to a target gene can cut DN as9 guide RN complex Resulting cut in target gene by as9 1 3 Normal (functional) gene for use as a template by repair enzymes YOPLSM (a) Scientists can disable ( knock out ) the target gene to study its normal function. (b) If the target gene has a mutation, it can be repaired. NULEUS Random nucleotides Normal nucleotides 5. herapeutic & Diagnostic echniques hapter Reading pp Embryonic stem cells ells generating all embryonic cell types Stem ells dult stem cells ells generating some cell types Stem cells are undifferentiated cells that can give rise to multiple differentiated cell types. ultured stem cells Different culture conditions Different types of differentiated cells Liver cells Nerve cells Blood cells Embryonic stem cells are considered more pluripotent than adult stem cells, though techniques are being developed to expand the potential of adult stem cell. 13

14 Stem ell herapy echniques are being developed to treat a person s own cells in vitro to produce a needed cell type which is then reintroduce into the patient to repair damaged tissue. 1 Remove skin cells from patient. Reprogram skin cells so the cells become induced pluripotent stem (ips) cells. Patient with damaged heart tissue or other disease 4 Return cells to patient, where they can repair damaged tissue. 3 reat ips cells so that they differentiate into a specific cell type. loned gene ene herapy Retrovirus capsid Bone marrow cell from patient 1 Insert RN version of normal allele into retrovirus. Viral RN Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. 3 Viral DN carrying the normal allele inserts into chromosome. Bone marrow stem cells can be genetically modified in vitro to fix a genetic defect and then reintroduced back into the patient. 4 Inject engineered cells into patient. Bone marrow Restriction Fragment Length Polymorphisms (RFLPs) Normal -globin allele Normal allele Sickle-cell allele 175 bp Sickle-cell mutant -globin allele 376 bp 01 bp Large fragment DdeI DdeI DdeI DdeI Large fragment DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of the -globin gene Large fragment 01 bp 175 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 376 bp DN from different sources cut with the same RE will result in different size DN fragments (RFLPs) 14

15 Short andem Repeat nalysis Many parts of the human genome contain short DN sequences repeated many times in a row (in tandem) (a) his photo shows Washington just before his release in 001, after 17 years in prison. the number of repeats in these regions is highly variable from person to person RFLP analysis can produce a DN fingerprint that is unique for each individual Source of sample Semen on victim Earl Washington Kenneth insley SR marker 1 17,19 16,18 17,19 SR marker 13,16 14,15 13,16 (b) hese and other SR data exonerated Washington and led insley to plead guilty to the murder. SR marker 3 1,1 11,1 1,1 EHNIQUE DN restriction enzyme Southern Blotting Restriction fragments I II III Nitrocellulose membrane (blot) el Heavy weight II Sickle-cell III Heterozygote allele I Normal -globin allele 1 Preparation of restriction fragments Sponge lkaline solution Paper towels el electrophoresis 3 DN transfer (blotting) Probe base-pairs I II III with fragments I II III Radioactively labeled probe for -globin gene Nitrocellulose blot 4 Hybridization with labeled probe Fragment from sickle-cell -globin allele Fragment from normal - globin allele Film over blot 5 Probe detection Key erms for hapter 0 recombinant DN, gene cloning, plasmid vector transformation, ligation, restriction enzymes genomic & cdn libraries, hybridization, probe gel electrophoresis, PR, R-PR dideoxynucleotide, RFLP, SR, SNP in situ hybridization, DN microarrays transgenic, stem cell, southern blot, RISPR-as9 in vivo, in vitro, in situ Relevant hapter Questions 1-10, 1 15