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Chapter 17. Application of Recombinant DNA Technology in Genetics The DNA Toolbox • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule. • Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes. • DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products. • To work directly with specific genes, scientists prepare genesized pieces of DNA in identical copies, a process called DNA cloning. DNA Cloning and Its Applications • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids. • Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome. • Cloned genes are useful for making copies of a particular gene and producing a protein product. • Gene cloning involves using bacteria to make multiple copies of a gene. • Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell. • Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA. • This results in the production of multiple copies of a single gene. DNA Cloning Bacterium 1 Gene inserted into Cell containing gene of interest plasmid Bacterial Plasmid chromosome Recombinant DNA (plasmid) Gene of interest 2 2 Plasmid put into bacterial cell Recombinant bacterium DNA of chromosome Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of Interest Copies of gene Basic research on gene Gene for pest resistance inserted into plants Protein harvested 4 Basic research and various applications Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats stunted growth Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites. • A restriction enzyme usually makes many cuts, yielding restriction fragments. • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other fragments. • DNA ligase is an enzyme that seals the bonds between restriction fragments. Restriction Enzymes Copyright © 2010 Pearson Education, Inc. Action of Restriction Enzymes Copyright © 2010 Pearson Education, Inc. Use of Restriction Enzymes Copyright © 2010 Pearson Education, Inc. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector. • A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there. Plasmid Vector Copyright © 2010 Pearson Education, Inc. Construction of Recombinant DNA Copyright © 2010 Pearson Education, Inc. Transformation Copyright © 2010 Pearson Education, Inc. Hummingbird cell TECHNIQUE Bacterial cell lacZ gene ampR gene Bacterial Restriction site Sticky ends Gene of interest Hummingbird DNA fragments plasmid Nonrecombinant plasmid Recombinant plasmids Bacteria carrying plasmids RESULTS Colony carrying nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones Selection of Recombinant DNA Copyright © 2010 Pearson Education, Inc. Storing Cloned Genes in DNA Libraries • A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome. • A genomic library that is made using bacteriophages is stored as a collection of phage clones. Foreign genome cut up with restriction enzyme or Recombinant phage DNA Bacterial clones (a) Plasmid library Recombinant plasmids Phage clones (b) Phage library BAC • A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert. • BACs are another type of vector used in DNA library construction. Large plasmid Large insert with many genes BAC clone (c) A library of bacterial artificial chromosome (BAC) clones YAC Copyright © 2010 Pearson Education, Inc. How big does a genomic library have to be to have a 95% or 99% chance of containing all the sequences in a genome? • The number of clones required to contain a genome depends on several factors: the average size of the cloned inserts, the size of the genome to be cloned, and the level of probability desired. • The number of clones in a library can be calculated as N = ln(1-P)/ln(1-f) N: the number of required clones P: the probability of recovering a given sequence f: the fraction of the genome in each clone Human Genomic Library • We wish to prepare a human genomic library large enough to have 99% chance of containing all the sequences in the genome. • If we construct the library using a plasmid vector with an average insert size of 5kb, more than 2.4 million clones would be required for a 99% probability of recovering any given sequence from the genome. • If the library is constructed in a YAC vector with an average insert size of 1Mb, then the library would only need to contain about 14,000 YACs. Question • An ampicillin-resistant, tetracycline-resistant plasmid, pBR322, is cleaved with PstI, which cleaves within the ampicillin-resistant gene. The cut plasmid is ligated with PstI-digested Drosophila DNA to prepare a genomic library, and the mixture is used to transform E. coli K12. 1. Which antibiotics should be added to the medium to select cells that have incorporated a plasmid? 2. What growth pattern should be selected to obtain plasmids containing Drosophila inserts? 3. How can you explain the presence of colonies that are resistant to both antibiotics? Copyright © 2010 Pearson Education, Inc. cDNA library • A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell. • A cDNA library represents only part of the genome — only the subset of genes transcribed into mRNA in the original cells. DNA in nucleus mRNAs in cytoplasm mRNA Reverse transcriptase Poly-A tail DNA Primer strand Degraded mRNA DNA polymerase cDNA cDNA Synthesis Copyright © 2010 Pearson Education, Inc. Screening a Library for Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene. • This process is called nucleic acid hybridization. • A probe can be synthesized that is complementary to the gene of interest. • For example, if the desired gene is 5¢ … G G C T A A C T T AG C … – Then we would synthesize this probe 3¢ C C G A T T G A A T C G 5¢ 3¢ Radioactive DNA probe Single-stranded DNA Mix with singlestranded DNA from genomic library Base pairing indicates the gene of interest · The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest. · Once identified, the clone carrying the gene of interest can be cultured. Hybridization is Used to Identify Similar DNA Sequences • Prepare library – Distribute libraries clones on petri dish – Transfer clones to nitrocellulose disk • Prepare probe – Previous cloned DNA – PCR fragment – Oligonucleotide • Screen library – Expose probe to clones on nitrocellulose – Determine location of matching clone by autoradiography or fluorescence Library Screening Copyright © 2010 Pearson Education, Inc. TECHNIQUE Radioactively labeled probe molecules Multiwell plates holding library clones Probe DNA Gene of interest Single-stranded DNA from cell Film • Nylon membrane Nylon Location of membrane DNA with the complementary sequence 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. • Kary Mullis (Chemistry 1993: invention of the polymerase chain reaction) • A three-step cycle — heating, cooling, and replication — brings about a chain reaction that produces an exponentially growing population of identical DNA molecules. 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 (denaturation), cooling (annealing), and replication (extension)—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules. Kary Mullis (1944-present): 1993 Nobel Prize in Chemistry TECHNIQUE Genomic DNA 1 2 5¢ 3¢ 3¢ Target sequence 5¢ Denaturation 5¢ 3¢ 3¢ 5¢ Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence PCR Process 5¢ TECHNIQUE 3¢ Target sequence Genomic DNA 3¢ 5¢ 1 Denaturation 5¢ 3¢ 3¢ 5¢ 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleotides Copyright © 2010 Pearson Education, Inc. Cycle 1 yields 2 molecules Genomic DNA 3¢ 5¢ 1 Heat to 3¢ 5¢ 5¢ 3¢ separate DNA strands 3¢ 5¢ 5¢ 2 Cool to allow primers to form hydrogen bonds with ends of target sequences Target sequence 3¢ 5¢ 3 DNA polymerase adds nucleotides to the 3¢ end of each primer 5¢ 5¢ 3¢ 5¢ 3¢ Primer 5¢ New DNA 3¢ Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules Gel Electrophoresis • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis. • This technique uses a gel as a molecular sieve to separate nucleic acids or proteins by size. • A current is applied that causes charged molecules to move through the gel. • Molecules are sorted into “bands” by their size. TECHNIQUE Power source Mixture of DNA molecules of different sizes Anode + – Cathode Gel 1 Power source – + Longer molecules 2 Shorter molecules Mixture of DNA fragments of different sizes Power source Longer (slower) molecules Gel Shorter (faster) molecules Completed gel Copyright © 2010 Pearson Education, Inc. Gel Electrophoresis and Hybridization to Map DNA Fragments • Southern blot – Cut whole genomic DNA with restriction enzyme – Separate DNA fragments by electrophoresis – Blot fragmented DNA to a filter – Hybridize to DNA probe – Observe matched bands by autoradiography or fluorescence Southern Blotting • A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization. • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel . Stain with Ethidium bromide Fig. 9.15 Copyright © 2010 Pearson Education, Inc. Radioactively labeled probe for b-globin gene I II III Probe base-pairs with fragments Fragment from sickle-cell b-globin allele Nitrocellulose blot Fragment from normal b-globin allele 4 Hybridization with radioactive probe I II III Film over blot 5 Probe detection Blot is removed washed, and exposed to X-ray film Copyright © 2010 Pearson Education, Inc. Use of RFLPs for Mapping – Single nucleotide polymorphism (SNP) is a variation at one base pair within a coding or noncoding sequence – Restriction fragment length polymorphism (RFLP) is a variation in the size of DNA fragments due to a SNP that alters a restriction site • In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis. • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene. • The procedure is also used to prepare pure samples of individual fragments. Normal b-globin allele 175 bp DdeI 201 bp DdeI Normal Sickle-cell allele allele Large fragment DdeI DdeI Large fragment Sickle-cell mutant b-globin allele 376 bp DdeI 201 bp 175 bp Large fragment 376 bp DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of b-globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles Restriction enzymes added DNA sample 1 DNA sample 2 w Cut z x Cut Cut y y Longer fragments z x Shorter fragments w y y STR site 1 STR site 2 Crime scene DNA Number of short tandem repeats match Suspect’s DNA Number of short tandem repeats do not match Crime scene DNA Suspect’s DNA (a) This photo shows Earl Washington just before his release in 2001, after 17 years in prison. Source of sample STR marker 1 STR marker 2 STR marker 3 Semen on victim 17, 19 13, 16 12, 12 Earl Washington 16, 18 14, 15 11, 12 Kenneth Tinsley 17, 19 13, 16 12, 12 (b) These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder. Question DNA samples were extracted from chimpanzee hair and used as templates for PCR. The primers used in our study flank highly polymorphic sites in human DNA that result from variable numbers of tandem nucleotide repeats. Several offspring and their putative parents were tested to determine whether the putative parents are real parents of the offspring. Copyright © 2010 Pearson Education, Inc. Question • The following partial restriction map shows a recombinant plasmid, pBIO220, formed by cloning a piece of Drosophila DNA (striped segment), including the rosy gene, into the vector pBR322, which also contains the penicillin-resistant gene. The vector part of the plasmid contains only the two E recognition sequences shown and no A or B sequences. The gel shows several restriction digests of pBIO220. Copyright © 2010 Pearson Education, Inc. Copyright © 2010 Pearson Education, Inc. 1. Use the stained gel pattern to deduce where restriction-enzyme recognition sequences are located in the cloned fragment. 2. A PCR-amplified copy of the entire 2000-bp rosy gene was used to probe a Southern blot of the same gel. Use the Southern blot results to deduce the location of rosy in the cloned fragment. Redraw the map showing the location of the rosy gene. Question • The gel shows the pattern of bands of fragments produced with several restriction enzymes. The enzymes used are identified above and below the gel. • One of the six restriction maps shown below is consistent with the pattern of bands shown in the gel. Copyright © 2010 Pearson Education, Inc. 1. From your analysis of the pattern of bands on the gel, select the correct map. 2. In a Southern blot prepared from this gel, the highlighted bands (pink) hybridized with the pep gene. Where is the pep gene located? DNA Sequencing • Relatively short DNA fragments can be sequenced by the dideoxy chain termination method. • Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths. • Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment. • The DNA sequence can be read from the resulting spectrogram. Copyright © 2010 Pearson Education, Inc. General Principals of Sanger Sequencing Method Fig. 9.17 Copyright © 2010 Pearson Education, Inc. Automated DNA sequencing TECHNIQUE DNA (template strand) Primer DNA polymerase Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) dATP ddATP dCTP ddCTP dTTP ddTTP dGTP ddGTP Copyright © 2010 Pearson Education, Inc. TECHNIQUE DNA (template strand) Labeled strands Shortest Direction of movement of strands Longest Longest labeled strand Detector Laser RESULTS Shortest labeled strand Last base of longest labeled strand Last base of shortest labeled strand Automated DNA Sequencing • Output from an automated DNA sequencing reaction – Each lane displays the sequence obtained from a separate DNA sample and primer. Sequencing Results • Computer reads of the sequence complementary to the template strand from right to left (5’ à 3’ direction). Machine generates complementary strand. Ambiguities are recorded as an “N” and can sometimes be resolved by a technician. Copyright © 2010 Pearson Education, Inc. Positional Cloning: Use of Polymorphic DNA Markers to Clone Genes via Linkage A pedigree of the royal family descended from Queen Victoria In which hemophilia A is segregating Positional Cloning: Step 1 • Find extended families in which disease is segregating. • Use panel of polymorphic markers spaced at 10 cM intervals across all chromosomes. – 300 markers total • Determine genotype for all individuals in families for each DNA marker. • Look for linkage between a marker and disease phenotype. • Once region of chromosome is identified, a high resolution mapping is performed with additional markers to narrow down region where gene may lie. Positional cloning: Step 2 (Identifying Candidate Genes) • Once region of chromosome has been narrowed down by linkage analysis to 1000 kb or less, all genes within are identified. • Candidate genes – Usually about 17 genes per 1000 kb fragment – Identify coding regions • Computational analysis to identify conserved sequences between species • Computational analysis to identify exon-like sequences by looking for codon usage, ORFs, and splice sites • Appearance on one or more EST clones derived from cDNA Computational Analysis of Genomic Sequences to Identify Candidate Genes Gene Expression Patterns Can Pinpoint Candidate Genes • Look in public database of EST sequences representing certain tissues. • Northern blot – RNA transcripts in the cells of a particular tissue (e.g., with disease) separated by electrophoresis and probed with candidate gene sequence Northern Blot Example of SRY Positional cloning: Step 3 • Find the gene responsible for the phenotype. – Expression patterns • RNA expression assayed by Northern blot or PCR amplification of cDNA with primers specific to candidate transcript • Look for misexpression (no expression, underexpression, overexpression) – Sequence differences • Missense mutations identified by sequencing coding region of candidate gene from normal and abnormal individuals – Transgenic modification of phenotype • Insert the mutant gene into a model organism. Transgenic Analysis Can Prove Candidate Gene Example: Positional Cloning of Cystic Fibrosis Gene • Linkage analysis places CF on chromosome 7 Northern blot analysis reveals only one of candidate genes is expressed in lungs and pancreas. -Every CF patient has a mutated allele of the CFTR gene on both chromosome 7 homologs. -Location and number of mutations indicated under diagram of chromosome -CFTR is a membrane protein. -TMD-1 and TMD-2 are transmembrane domains. Proving CFTR is the Right Gene • Phenotype eliminates gene function. – Cannot use transgenic technology. • Instead perform CFTR gene “knockout” in mouse to examine phenotype without CFTR gene. – Targeted mutagenesis • Introduce mutant CFTR into mouse embryonic cells in culture. • Rare double recombinant events with homologous wild-type CFTR gene are selected for. • Mutant cell is introduced into normal mouse embryos where they incorporate into germ line. • Knockout mouse created.