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Ch. 14: DNA Technology
Recombinant DNA Technology = techniques for recombining genes from different sources in
vitro and transferring the recombinant DNA into a cell where it may be expressed

Allows genes to be moved across the species barrier

Goal: Improvement of human health and food production
DNA CLONING (Fig. 20.1)
Requires:
1) Restriction Enzymes = enzymes that recognize short, specific nucleotide sequences called
recognition sequences or restriction sites.
o Restriction = process in which (foreign) DNA is cut into smaller segments

Only cut at specific points within those sequences = Recognition
Sequences

Occur naturally in bacteria (to defend against phages)
o Several hundred restriction enzymes exist that use about 100 different specific
recognition sequences
o Recognition sequences are symmetric = have same sequence of 4-8 nucleotides
on both strands, but they run in opposite directions
o Restriction enzymes usually cut both strands of DNA in a staggered manner,
resulting in double-stranded fragments with single-stranded ends = sticky ends

Some may cut straight through
o Sticky ends of restriction fragments are used in the lab to join DNA pieces from
different sources

These unions are made permanent by the addition of DNA ligase
o Outcome: Recombinant DNA (rDNA): a DNA molecule carrying a new
combination of genes
2) Cloning Vector = a DNA molecule that can carry foreign DNA into a cell and replicate
there

Most common ones are bacterial plasmids and viruses

Plasmid = small circles of DNA that carry extra genes in bacteria (“optional”)
o Restriction fragments of foreign DNA can be spliced into a bacterial plasmid
without interfering with its ability to replicate within the bacterial cell.

Done by cutting both with SAME restriction enzyme  identical
sticky ends
o Isolated recombinant plasmids can be introduced into bacterial cells by
transformation (recall Griffith  bacteria can take up naked pieces of DNA
from their surroundings, including plasmids).

Bacteriophages (viruses) can also be used as vectors
o The middle of the linear genome, which contains nonessential genes, is
deleted by using restriction enzymes
o Restriction fragments of foreign DNA are then inserted
o Recombinant phage DNA is introduced into an E. coli cell  Transduction
o Phage replicates itself inside the bacterial cell

Sometimes it is necessary to clone DNA in eukaryotic cells rather than in bacteria, in
which case yeast and animal cells may be used.
PROCEDURE FOR CLONING A EUKARYOTIC GENE IN A BACTERIAL PLASMID
(Figure 20.3 – Learn it, know it, love it!!!)
Step 1: Isolation of vector and gene-source DNA

Ex. Foreign DNA is human and plasmid from E. coli has two genes:
o ampR = antibiotic resistance to ampicillin
o lacZ = codes for β-galactosidase (think “lac operon”)

Note: the recognition sequence for the restriction enzyme is within the
lacZ gene
Step 2: Insertion of Gene-Source DNA into the vector

Digestion = restriction enzyme cuts plasmid DNA at the restriction site, disrupting the
lacZ gene

Foreign DNA is cut into thousand of fragments by the same restriction enzyme
o ONE of these fragments contains the gene of interest
o Methods for detecting the DNA of a gene depends directly on base pairing
between the gene if interest and a complementary sequence on another nucleic
acid molecule, a process called nucleic acid hybridization (“Southern Blotting)

The complementary molecule, a short piece of RNA or DNA is called a
nucleic acid probe (usually radioactive)

Produces identical sticky ends on both foreign DNA and the plasmid

Foreign DNA fragments are mixed

Some lacZ gene sticky ends may match up with sticky ends on foreign DNA, some will
re-seal on their own opposite sticky ends

Addition of DNA Ligase seals sticky ends together
Step 3: Introduction of Cloning Vector into Bacterial Cells

Naked DNA is added to a bacterial culture

Some bacteria will take up the plasmid DNA by transformation
Step 4: Cloning of Cells (and the foreign DNA)

Bacteria with the recombinant plasmid will be able to reproduce on the medium
containing ampicillin, cloning the inserted gene in the process
Step 5: Identification of Cell Clones Carrying the Gene of Interest

X-gal turns blue when hydrolyzed by β-galactosidase, so it can be used as an indicator of
which cells have been transformed by plasmids containing the foreign DNA.
o Since the foreign DNA insert disrupts the lacZ gene, bacterial colonies that have
successfully acquired the foreign DNA fragment will be white. Those that lack
the foreign gene will turn blue (because the lacZ gene will be fully functional.)
Cloning and Expressing Eukaryotic Genes: Problems and Solutions
Problem: How to turn the gene “on”
Solution: Insert foreign DNA into an inducible operon
Problem: Eukaryotic DNA fragments of interest often contain introns
Solution: Make artificial eukaryotic genes that lack introns (Fig. 20.5)

cDNA = DNA that is built by addition of an enzyme called reverse transcriptase to the
mRNA for a gene of interest (post mRNA processing)
o Reverse Transcriptase = builds a strand of DNA from mRNA (backwards from
RNA polymerase)
Problem: Prokaryotic cells are not capable of making post-translational modifications required
for some eukaryotic proteins (no E.R. or Golgi to add lipid or carbohydrate groups)
Solution: Use eukaryotic cells, like yeast, animal, or plant cells

Aggressive techniques for inserting foreign DNA into eukaryotic cells:
o Electroporation = a brief electric pulse applied to a cell solution causes temporary
holes in the plasma membrane, through which the DNA can enter
o Thin needles can inject DNA directly into a eukaryotic cell
o DNA Gun = DNA is attached to microscopic metal particles that can be fired into
plant cells with a gun