Chapter 14 The Techniques of Molecular Genetics © John Wiley & Sons, Inc.

Chapter Outline Basic Techniques used to Identify, Amplify, and Clone Genes Construction and Screening of DNA Libraries The Molecular Analysis of DNA, RNA, and Protein The Molecular Analysis of Genes and Chromosomes © John Wiley & Sons, Inc.

Basic Techniques Used to Identify, Amplify, and Clone Genes Recombinant DNA, gene cloning, and DNA amplification techniques allow scientists to isolate and characterize essentially any DNA sequence from any organism. © John Wiley & Sons, Inc.

Gene Cloning Gene cloning is the isolation and amplification of a given gene. A recombinant DNA molecule is a DNA molecule made by joining two or more different DNA molecules. © John Wiley & Sons, Inc.

Amplification of a Gene In Vivo A minichromosome carrying the gene of interest is produced in the test tube. The recombinant minichromosome is introduced into a host cell (such as E. coli), and the host cell replicates the minochromosome. © John Wiley & Sons, Inc.

Amplification of a Gene In Vitro Short DNA strands complementary to DNA sequences on either side of the gene of interest are synthesized. These short DNA strands are used to initiate the amplification of the gene by a heat-stable DNA polymerase in the polymerase chain reaction (PCR). © John Wiley & Sons, Inc.

Restriction Endonucleases Restriction endonucleases make site-specific cuts in DNA. The nucleotide sequences are called restriction sites. Restriction endonucleases protect bacteria from foreign DNA. Bacteria protect endogenous restriction sites by methylation. Restriction enzymes commonly recognize palindromic sequences. © John Wiley & Sons, Inc.

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© John Wiley & Sons, Inc.

Structure of an EcoRI-DNA Complex © John Wiley & Sons, Inc.

Many Restriction Endonucleases Make Staggered Cuts © John Wiley & Sons, Inc.

When DNA is cleaved with a restriction endonuclease that makes staggered cuts, all of the resulting restriction fragments have complementary single-stranded termini. The complementary single-stranded termini can hydrogen bond with each other and be joined together by DNA ligase. © John Wiley & Sons, Inc.

Construction of Recombinant DNA Molecules In Vitro © John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

Plasmid Vectors Circular, double-stranded circular DNA molecules present in bacteria. Range from 1 kb to over 200 kb. Replicate autonomously. Many carry antibiotic-resistance genes, which can be used as selectable markers. Many useful cloning vectors were derived from plasmid pBR322. © John Wiley & Sons, Inc.

Bacteriophage Vectors Most bacteriophage cloning vectors have been constructed from the phage  chromosome. The central one-third (about 15 kb) of the  chromosome contains genes required for lysogeny but not for lytic growth. This portion of the chromosome can be excised and replaced with foreign DNA. The foreign DNA inserted must be 10-15 kb. © John Wiley & Sons, Inc.

Cosmid Vectors Hybrids between plasmids and the phage  chromosome. Replicate autonomously in E. coli. Can be packaged in vitro into phage  heads. Accept inserts of 35-45 kb. © John Wiley & Sons, Inc.

Phagemid Vectors Contain components from phage chromosomes and plasmids. Replicate in E. coli as double-stranded plasmids. Addition of a helper phage causes the phagemid to switch to the phage mode of replication, resulting in the packaging of single-stranded DNA into phage heads. © John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

The Blue-White Color Test The E. coli lacZ gene encodes -galactosidase. -galactosidase converts the colorless substrate Xgal into a blue product. Cells with -galactosidase activity produce blue colonies when grown on Xgal; cells lacking -galactosidase activity produce white colonies. © John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

Eukaryotic and Shuttle Vectors Because different organisms use different origins of replication and regulatory signals, different cloning vectors must be used in different species. Special cloning vectors can replicate in other prokaryotes and in eukaryotes. Shuttle vectors can replicate in E. coli and in another species. © John Wiley & Sons, Inc.

Yeast Artificial Chromosomes (YACs) Genetically engineered yeast minichromosomes. Accept foreign DNA inserts of 200-500 kb. Contain a yeast origin of replication, yeast centromere, two yeast telomeres, a selectable marker, and a polycloning site. © John Wiley & Sons, Inc.

BACs and PACs Bacterial artificial chromosomes (BACs) have been constructed from bacterial fertility (F) factors. Bacteriophage P1 artificial chromosomes (PACs) have been constructed from bacteriophage P1 chromosomes. BACs and PACs accept 150-300 kb inserts and are less complex than YACs. © John Wiley & Sons, Inc.

The Polymerase Chain Reaction (PCR) Synthetic nucleotides complementary to known flanking sequences are used to prime enzymatic amplification of the sequence of interest. Three repeated steps Denaturation of genomic DNA (92-95°C) Annealing of denatured DNA to oligonucleotide primers (50-60°C) Replication of the DNA segment between the sites complementary to the primers (70-72°C) Amplification occurs exponentially; each cycle doubles the number of molecules of the sequence of interest. © John Wiley & Sons, Inc.

Taq Polymerase DNA polymerase from Thermus aquaticus is used for PCR because it is heat-stable. Taq polymerase lacks proofreading activity, so errors are introduced into the amplified DNA at low but significant frequencies. When high fidelity is required, heat-stable polymerases with proofreading activity are used (Pfu or Tli). Taq is amplifies fragments of DNA larger than a few thousand base pairs inefficiently. For amplification of long segments of DNA (up to 35 kb), Tfl polymerase is used. © John Wiley & Sons, Inc.

Applications of PCR Diagnosis of inherited human diseases (e.g., prenatal diagnosis). Identification of individuals in forensic cases from small DNA samples. © John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

Key Points The discovery of restriction endonucleases—enzymes that recognize and cleave DNA in a sequence-specific manner—allowed scientists to produce recombinant DNA molecules in vitro. DNA sequences can be inserted into small, self-replicating DNA molecules called cloning vectors and amplified by replication in vivo after being introduced into living cells by transformation. © John Wiley & Sons, Inc.

Key Points The polymerase chain reaction (PCR) can be used to amplify specific DNA sequences in vitro. © John Wiley & Sons, Inc.

Construction and Screening of DNA Libraries DNA libraries can be constructed and screened for genes and other sequences of interest. © John Wiley & Sons, Inc.

DNA Libraries A genomic DNA library is a set of DNA clones collectively containing the entire genome of an organism. A cDNA library contains the coding regions of the expressed genes of an organism. It is made of complementary DNA (cDNA) synthesized from RNA by reverse transcriptase. © John Wiley & Sons, Inc.

Cloning Restriction Fragments with Complementary Single-Stranded Ends © John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

Amplification of Recombinant DNA Antibiotic-sensitive recipient cells are transformed with the recombinant DNA molecule. Transformed cells are selected by growth under conditions requiring the presence of a selectable marker present on the recombinant DNA molecule (usually an antibiotic). The recombinant DNA molecule is amplified by the host cell. © John Wiley & Sons, Inc.

Synthesis of Double-Stranded cDNAs from mRNA © John Wiley & Sons, Inc.

Screening DNA Libraries for Genes of Interest Genetic Selection—searching for a DNA sequence that restores the wild-type phenotype to a mutant organism. Molecular hybridization is based on the hybridization of similar DNA sequences. © John Wiley & Sons, Inc.

Colony Hybridization © John Wiley & Sons, Inc.

Key Points DNA libraries can be constructed that contain complete sets of genomic DNA sequences or DNA copies (cDNAs) of mRNAs in an organism. Specific genes or other DNA sequences can be isolated from DNA libraries by genetic complementation or by hybridization to labeled nucleic acid probes containing sequences of known function. © John Wiley & Sons, Inc.

The Molecular Analysis of DNA, RNA, and Protein DNA, RNA, or protein molecules can be separated by gel electrophoresis, transferred to membranes, and analyzed by various procedures. © John Wiley & Sons, Inc.

Analysis of DNAs by Southern Blot Hybridization DNA molecules can be separated by size by gel electrophoresis using agarose or acrylamide gels for larger and small DNA molecules, respectively. DNA molecules can then be transferred from the gel onto a nitrocellulose or nylon membrane using a technique called a Southern blot. DNA on the membrane can be hybridized with DNA probes. © John Wiley & Sons, Inc.

Agarose Gel Electrophoresis © John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

Southern Blot: Transferring DNA from the Gel to a Membrane © John Wiley & Sons, Inc.

Identification of a Specific Fragment by Southern Blot Hybridization © John Wiley & Sons, Inc.

Detection of Wild-Type and Mutant Alleles of the Cystic Fibrosis Gene © John Wiley & Sons, Inc.

Analysis of RNAs by Northern Blot Hybridizations The Northern Blot procedure is nearly identical to Southern blotting, except RNA is sensitive to degradation by RNases; contamination with these enzymes must be prevented. RNA molecules contain extensive secondary structure and must be kept denatured during electrophoresis. Northern blots are useful in studies of gene expression. © John Wiley & Sons, Inc.

Northern Blot Hybridization Data (RNA from roots, leaves, and flowers of A. thaliana) © John Wiley & Sons, Inc.

Analysis of RNAs by Reverse Transcriptase-PCR (RT-PCR) © John Wiley & Sons, Inc.

Analysis of Proteins by Western Blot Techniques Polypeptides are separated by polyacrylamide gel electrophoresis in the presence of a detergent that denatures the proteins. Proteins are transferred from the gel to a nitrocellulose membrane. Individual proteins are detected with antibodies. © John Wiley & Sons, Inc.

Key Points DNA restriction fragments and other small DNA molecules can be separated by agarose or acrylamide gel electrophoresis and transferred to nylon membranes to produce DNA gel blots called Southern blots. The DNAs on Southern blots can be hybridized to labeled DNA probes to detect sequences of interest by autoradiography. © John Wiley & Sons, Inc.

Key Points When RNA molecules are separated by gel electrophoresis and transferred to membranes for analysis, the resulting RNA gel blots are called northern blots. RNA molecules can be detected and analyzed by reverse transcriptase-PCR (RT-PCR). © John Wiley & Sons, Inc.

Key Points When proteins are transferred from gels to membranes and detected with antibodies, the products are called western blots. © John Wiley & Sons, Inc.

The Molecular Analysis of Genes and Chromosomes The sites at which restriction enzymes cleave DNA molecules can be used to construct physical maps of the molecules; however, nucleotide sequences provide the ultimate physical maps of DNA molecules. © John Wiley & Sons, Inc.

Mapping Restriction Enzyme Cleavage Sites © John Wiley & Sons, Inc.

Restriction maps reflect true physical distances (unlike genetic maps). Restriction maps can be combined with other molecular techniques to construct physical maps of entire genomes. © John Wiley & Sons, Inc.

Techniques Necessary for Sequencing DNA Restriction enzymes to prepare homogenous samples of specific segments of chromosomes. Gel electrophoresis procedures able to resolve DNA fragments differing in length by a single nucleotide. Gene-cloning techniques allowing preparation of large quantities of a DNA molecule. Sanger sequencing Technique is used to determine nucleotide sequences. © John Wiley & Sons, Inc.

DNA Sequencing A population of DNA fragments is generated. One end is common to all fragments (the 5’ end of the sequencing primer). The other end terminates at all possible positions (the 3- terminus). © John Wiley & Sons, Inc.

2',3'-Dideoxyribonucleoside Triphosphates © John Wiley & Sons, Inc.

Automated DNA Sequencing Fluorescent dyes are used for detection of DNA chains instead of radioactive isotopes. Products of all four chain terminator reactions are separated through a single gel or capillary tube. Photocells detect fluorescence of the dyes as they pass through the gel or capillary tube. Output of the photocell is directly transferred to a computer for analysis. © John Wiley & Sons, Inc.

Automated Sanger DNA Sequencing © John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

© John Wiley & Sons, Inc.

Key Points Detailed physical maps of DNA molecules can be prepared by identifying the sites that are cleaved by various restriction endonucleases. The nucleotide sequences of DNA molecules provide the ultimate physical maps of genes and chromosomes. © John Wiley & Sons, Inc.