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Part 4: Genomic Analysis Recombinant DNA Technology Review
  1. Recombinant DNA Technology Combines Several Experimental Techniques
     
     
     
     
    Recombinant DNA refers to a combination of DNA molecules, usually from different biological sources, that are not found together in nature.
     
     
     
     
    The basic procedure for producing recombinant DNA involves generating specific DNA fragments using restriction enzymes, joining these fragments with a vector, and transferring the recombinant DNA molecule to a host cell to produce many copies that can be recovered from the host cell.
  2. Recombinant DNA Technology Is the Foundation of Genome Analysis
     
     
     
     
    Through recombinant DNA technology, researchers can investigate many aspects of gene organization, gene function, and factors that regulate gene expression.
  3. Restriction Enzymes Cut DNA at Specific Recognition Sequences
     
     
     
     
  4. A enzyme binds to DNA at a specific recognition sequence and cleaves the DNA to produce restriction fragments.
     
     
     
     
  5. Most recognition sequences are , and restriction enzymes often cleave these sequences in an offset manner (Figure 19.2 and Figure 19.3 ).
     
     
     
     
    DNA ligase joins restriction fragments covalently to produce intact DNA molecules ( Figure 19.4).
     
     
     
     
  6. Vectors Carry DNA Molecules to Be Cloned Vectors are carrier DNA molecules that can replicate cloned DNA fragments in a host cell. Vectors must be able to replicate independently and should have several restriction enzyme sites to allow insertion of a DNA fragment.
     
     
     
     
    A plasmid is an extrachromosomal double-stranded DNA molecule that replicates autonomously in bacterial cells ( Figure 19.5).
     
     
     
     
    Plasmids used for DNA cloning usually have been engineered to contain a number of convenient restriction sites and a marker gene to select for its presence in the host cell ( Figure 19.6 7).
     
     
     
     
    The central third of lambda (λ) phage vectors can be replaced with foreign DNA without affecting the ability to infect cells and replicate. Lambda vectors can carry up to 20 kb of cloned DNA ( Figure 19.9 and Figure 19.10).
     
     
     
     
    Cosmid vectors are created by combining parts of l phage and parts of plasmids. Cosmids contain the cos sites of lambda, which are necessary for packaging of phage DNA into phage particles. Once inside the bacterial cell, cosmids replicate as plasmids. Cosmids can carry almost 50 kb of inserted DNA.
     
     
     
     
    Bacterial artificial chromosomes (BACs) are based on F factor and can carry up to 300 kb of inserted DNA (Figure 19.11).
     
     
     
     
    Expression vectors are engineered to express a gene of interest to produce large quantities of the encoded protein (Figure 19.12). DNA_cloning
     
     
     
     
  7. Yeast Cells Are Used as Eukaryotic Hosts for Cloning Yeast is widely used as a host for DNA cloning because it can be grown easily, its genetics have been studied intensively, its genome has been sequenced, it can posttranslationally modify eukaryotic proteins, and it is considered to be safe.
     
     
     
     
    Yeast artificial chromosomes (YACs) can contain 100 - 1000 kb of inserted DNA (Figure 19.14).
     
     
     
     
  8. Genes Can Be Transferred to Eukaryotic Cells In addition to bacteria and yeast, plant and animal cells can serve as hosts for recombinant DNA.
     
     
     
     
    Agrobacterium tumefaciens can be used to transform plant cells with T-DNA containing foreign DNA (Figure 19.15).
     
     
     
     
    The T-DNA and insert integrate into the plant genome. The plant cells can be grown in tissue culture and eventually regenerate a mature plant carrying a foreign gene.
     
     
     
     
    DNA can be transferred to mammalian cells by several methods, including endocytosis and encapsulation in liposomes followed by fusion with cell membranes.
     
     
     
     
    Transgenic mice can be produced by transferring YACs by microinjection into the nucleus of a mouse oocyte ((Figure 19.16).
     
     
     
     
  9. The Polymerase Chain Reaction Makes DNA Copies without Host Cells
     
     
     
     
    The polymerase chain reaction ( PCR ) copies a specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small quantities.
     
     
     
     
    PCR requires two oligonucleotide primers, one complementary to the 3' end of one strand of the DNA to be amplified and one complementary to the 3' end of the other strand.
     
     
     
     
    The primers anneal to denatured DNA, and the complementary strands are synthesized by a heat-stable DNA polymerase (Figure 19.17).
     
     
     
     
    The three steps of PCR - denaturation, primer annealing, and extension - are repeated over and over to amplify the DNA exponentially. chromosome-sorting pulsed-field-gel-electrophoresis
  10. Libraries Are Collections of Cloned Sequences A genomic library contains at least one copy of all the sequences in the genome of interest. Genomic libraries are constructed by cutting genomic DNA with a restriction enzyme and ligating the fragments into vectors. The choice of vector usually depends on the size of the genome.
     
     
     
     
    The number of clones in a library needed to give a certain probability of containing all genomic sequences is calculated as N = ln(1 � P)/ln(1 � f), where N is the number of required clones, P is the probability of recovering a sequence, and f is the fraction of the genome in each clone.
     
     
     
     
    Libraries can be made from subgenomic fractions such as a single chromosome.
     
     
     
     
    A cDNA library contains complementary DNA copies made from the mRNAs present in a cell population and represents the genes that are transcriptionally active at the time the cells were collected for mRNA isolation.
     
     
     
     
    A cDNA library is prepared by isolating mRNA from cells, synthesizing the complementary DNA using reverse transcriptase, and cloning the cDNA molecules into a vector (Figure 19.20).
     
     
     
     
    Reverse transcriptase PCR (RT-PCR) can be used to generate cDNA from mRNA by first making a single-stranded cDNA copy of the mRNAs using reverse transcriptase and then using PCR to copy the single-stranded DNA into double-stranded DNA.
  11. Specific Clones Can Be Recovered from a Library Probes complementary to part of a gene are used to screen a library to recover clones of a specific gene.
     
     
     
     
    To screen a plasmid library, clones from the library are grown on agar plates to produce colonies. The colonies are screened by transferring bacterial colonies from the plate to a filter and hybridizing the filter with a nucleic acid probe to the DNA sequence of interest (Figure 19.21).
     
     
     
     
    The colony corresponding to the one the probe identified on the filter is identified and recovered.
     
     
     
     
    A phage library is screened by plaque hybridization.
  12. Cloned Sequences Can Be Characterized in Several Ways A restriction map establishes the number and order of restriction sites and the distance between restriction sites on a cloned DNA segment ( Figure 19.22 and Figure 19.23).
     
     
     
     
    A Southern blot is used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments from restriction digest.
     
     
     
     
    Southern blots can also be used to determine whether a clone contains all or part of a gene and to ascertain the size and sequence organization of a gene or DNA sequence of interest ( Figure 19.24 and Figure 19.25).
     
     
     
     
  13. DNA Sequencing Is the Ultimate Way to Characterize a Clone
     
     
     
     
    The most common method of DNA sequencing is dideoxy chain termination sequencing, developed by Sanger ( Figure 19.26 and Figure 19.27).
     
     
     
     
    Large-scale genome sequencing is automated and uses fluorescent dye�labeled dideoxynucleotides ( Figure 19.28 and Figure 19.29). 
     
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
    
 
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