Chapter 20 Notes
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Recombinant DNA—DNA in which nucleotide sequences from two different sources (often different species) are
combined into the same DNA molecule
Genetic engineering: the direct manipulation of genes for practical purposes
o Ex: manufacture of hundreds of protein products, such as hormones and blood-clotting factors
Using DNA technology, scientists can make recombinant DNA and then introduce it into cultured cells that
replicate the DNA and express its genes, yielding a desired protein
Biotechnology: manipulation of organisms or their components to make useful products
o Ex: use of microbes to make wine and cheese; selective breeding; exchanging of genes between
organisms
20.1
DNA cloning permits production of multiple copies of a specific gene or other DNA segment
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Naturally occurring DNA molecules are very long, and a single molecule usually carries many genes
o Genes may occupy only a small proportion of the chromosomal DNA, the rest being noncoding
nucleotide sequences
Gene cloning: preparing well-defined, gene-sized pieces of DNA in multiple identical copies
DNA Cloning and Its Applications: A Preview
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Most methods for cloning pieces of DNA in the lab share certain general features
o Common approach uses bacteria and plasmids
For cloning genes in the lab, a plasmid is first isolated from a bacterial cell, and then the foreign DNA is inserted
into it
o Resulting is now a recombinant DNA molecule
o Plasmid is returned to a bacterial cell, producing a recombinant bacterium, which reproduces to form a
clone of identical cells
Cloned genes are useful for two basic purposes
o Make many copies of particular gene
o Produce protein product
Most protein-coding genes exist in only one copy per genome so the ability to clone such rare DNA fragments is
extremely valuable
Using Restriction Enzymes to Make Recombinant DNA
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Gene cloning and genetic engineering made possible by discovery of enzymes that cut DNA molecules at a
limited number of specific locations
o Called restriction enzymes (restriction endonucleases)
 Protect the bacterial cell against intruding DNA from other organisms
 Work by cutting up the foreign DNA
 Process called restriction
Hundreds of different restriction enzymes have been identified and isolated
o Each very specific
 Recognizes short DNA sequence called restriction site
 Cuts both DNA strands at specific points within this restriction site
Most restriction enzymes recognize sequences containing 4-8 nucleotides
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Any sequence this short usually occurs by chance many times in a long DNA molecule, yielding a set of
restriction fragments
 A restriction enzyme cuts a DNA molecule in a reproducible way
Most useful restriction enzymes cleave the sugar-phosphate backbones in both DNA strands in a staggered way
o Resulting double-stranded restriction fragments have at least one single-stranded end called a sticky
end
 Form hydrogen-bonded base pairs with complementary sticky ends on any other DNA molecules
cut with the same enzyme
 Temporary but can be made permanent by DNA ligase
Cloning a Eukaryotic Gene in Bacterial Plasmid
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Cloning vector: original plasmid; defined as a DNA molecule that can carry foreign DNA into a cell and replicate
there
o Why plasmids?
 Can be easily isolated
 Easily manipulated to form recombinant plasmids by insertion of foreign DNA
 Easily reintroduced into bacterial cells
Producing Clones of Cells
1. Isolate bacterial plasmids from E. coli and DNA containing the gene of interest from human cells grown
in laboratory culture;
2. Both plasmid and human DNA are digested with the same restriction enzyme, one that produces stick
yenze; cuts the plasmid DNA at its single restriction site within the lacZ gene; cuts human DNA at
multiple sites
3. Mix the human DNA fragments with the cut plasmids, allowing base pairing between their
complementary sticky ends; then add DNA ligase;
4. DNA prepared in last step mixed with bacteria that have a mutation in their own lacZ gene, making them
unable to hydrolyze lactose
5. In cloning, bacteria are plated out on solid nutrient medium containing ampicillin and X gal
 How do we know the cell clones carrying recombinant plasmids?
 First, only cells with a plasmid will reproduce, because amp gene makes them resistant
to ampicillin in the medium
 Second, color of the colonies allows us to distinguish colonies of bacteria with
recombinant plasmids form those with nonrecombinant plasmids
Identifying Clones Carrying a Gene of Interest
o To screen all the colonies with recombinant plasmids for a clone of cells containing a gene of interest,
look at the gene itself or for its protein product
 Look at gene itself:
 DNA is detected by its ability to base-pair with a complementary sequence on another
nucleic acid molecule
o Called nucleic acid hybridization
 Complementary molecule of either RNA or DNA is called nucleic acid probe
o Each probe molecule with hydrogen-bond to a complementary strand in the
desired gene will be labeled with a radioactive isotope
 Key step is denaturation: separation of its two strands
o Happens with chemicals or heat
Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
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DNA cloning in cells remains the best method for preparing large quantities of a particular gene or other DNA
sequence
o When the source of DNA is impure, PCR is quicker and more selective
 In this technique, any specific target segment within one or many DNA molecules can be quickly
amplified in a test tube
 With automation, PCR can make billions of copies of target segment of DNA in a few hours
In PCR procedure, three step cycle brings about chain reaction that produces growing population of identical
DNA molecules
o During each cycle, reaction mixture is heated to denature the DNA strands and then cooled to allow
annealing of short, single-stranded DNA primers complementary to sequences on opposite strands at
each end of the target sequence
o Finally, a heat-stable DNA polymerase extends the primers in the 5’  3’ direction
Key to automated PCR was the discovery of an unusual heat-stable dNA polymerase
Only minute amounts of DNA need to be present in the starting material to use PCR
PCR cannot substitute for gene cloning in cells when large amounts of a gene are desired
o Occasional errors during PCR replication impose limits on the number of good copies that can be made
by this method
PCR used to make enough of a specific DNA fragment to clone it merely by inserting it into a vector
PCR used to amplify DNA from wide variety of sources
o Fragments of ancient DNA
o DNA from fingerprints or from tiny amounts of blood, tissue, or semen found at crime scenes
o DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders
o DNA of viral genes from cells infected with viruses that are difficult to detect