Chapter 12 Recombinant DNA: Cloning and Creation of Chimeric Genes

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Chapter 12Recombinant DNA Cloning and Creation
of Chimeric Genes
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Outline

• What does it mean to clone ?
• What is a plasmid ?
• What is a DNA library ?
• What is Southern Blotting ?
• What is the polymerase chain reaction (PCR) ?

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12.1 What Does It Mean To Clone?

• Clone a collection of molecules or cells, all
  identical to an original molecule or cell
• To "clone a gene" is to make many copies of it -
  for example, in a population of bacteria
• Gene can be an exact copy of a natural gene
• Gene can be an altered version of a natural gene
• Recombinant DNA technology makes it possible

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Plasmids Are Very Useful in Cloning Genes

• Plamids are naturally occurring extrachromosomal
  DNA.
• Plasmids are circular dsDNA.
• Plasmids can be cleaved by restriction enzymes,
  leaving sticky ends.
• Artificial plasmids can be constructed by linking
  new DNA fragments to the sticky ends of plasmid.
• These recombinant molecules can be autonomously
  replicated, and hence propagated.

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Virtually Any DNA Sequence Can Be Cloned
Nuclease cleavage at a restriction site
linearizes the circular plasmid so that a foreign
DNA fragment can be inserted. Recombinant
plasmids are hybrid DNA molecules consisting of
plasmid DNA sequences plus inserted DNA elements
(pink here). Figure 12.2 An EcoRI restriction
fragment of foreign DNA can be inserted into a
plasmid.
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Chimeric Plasmids

• Named for mythological beasts with body parts
  from several creatures
• Such hybrid molecules are called chimeric
  plasmids.
• After cleavage of a plasmid with a restriction.
  enzyme, a foreign DNA fragment can be inserted
• Ends of the plasmid/fragment are closed with DNA
  ligase to form a "recombinant plasmid".
• Plasmid can replicate when placed in a suitable
  bacterial host.
• See Figure 12.2.

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Cloning Vectors

• Cloning vectors are plasmids that can be modified
  to carry new genes
• Plasmids useful as cloning vectors must have
• a replicator (origin of replication).
• a selectable marker (antibiotic resistance gene).
• a cloning site (site where insertion of foreign
  DNA will not disrupt replication or inactivate
  essential markers).

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12.2 What is a DNA Library?

• A DNA library is a set of cloned DNA fragments
  that together represent the genes of a particular
  organism
• Any particular gene may represent a tiny, tiny
  fraction of the DNA in a given cell.
• So, it can't be isolated directly.
• The trick is to find the fragment or fragments in
  the library that contain the desired gene.

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Identifying Specific DNA Sequences by Southern
Blotting

• Finding one particular DNA segment among a vast
  population of different DNA fragments (e.g., in a
  genomic DNA preparation) is to exploit its
  sequence specificity to identify it.
• Southern blots (invented by E.M. Southern) do
  this.
• DNA fragments (the library) are fractionated by
  size with agarose gel electrophoresis.
• Gel is blotted to an absorbent support and then
  incubated with radioactively labeled
  oligonucleotide probes.
• An autoradiograph shows the hybridized DNA
  fragments.

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Identifying Specific DNA Sequences by Southern
Blotting
The Southern blotting technique involves the
transfer of electrophoretically separated DNA
fragments to an absorbent sheet and subsequent
detection of the specific DNA sequences.
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The Human Genome Project

• A working draft of the human genome was completed
  in June, 2000 and published in February 2001.
• The genomes of many other organisms have now been
  sequenced as well.
• Information about whole genome sequences has
  created a new branch of science called
  bioinformatics.

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12.4 What Is the Polymerase Chain Reaction (PCR)?

• What if you don't have enough DNA for colony
  hybridization or Southern blots?
• The small sample of DNA serves as template for
  DNA polymerase.
• Make complementary primers.
• Add primers in more than 1000-fold excess.
• Heat to make ssDNA, then cool.
• Run DNA polymerase (usually Taq).
• Repeat heating, cooling, polymerase cycle.

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12.4 What Is the Polymerase Chain Reaction (PCR)?
Figure 12.18 Polymerase chain reaction (PCR).
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End Chapter 12Recombinant DNA Cloning and
Creation of Chimeric Genes