Updated Summer 2015
Jerald D. Hendrix
A.
Recombinant DNA Technology
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Restriction Endonucleases
Creating a Recombinant DNA Library
Properties of a Cloning Vector
Screening a Recombinant DNA Library
DNA Sequencing
Polymerase Chain Reaction
Bioinformatics
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Type II Restriction Endonuclease
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Recognizes a specific sequence (recognition sequence) on
double stranded DNA, and . . .
Cleaves the DNA molecule at the recognition site
This makes Type II restriction endonucleases a very
specific and precise molecular scissors to cut DNA.
Recognition sequences are 4 – 8 nucleotide base pairs in
length, with 6 bp sequences the most common
Several hundred restriction endonucleases have been
discovered
Unless otherwise specified, the term “restriction
endonuclease” implies “Type II”
The term “restriction enzyme” is also used synonymously
with “Type II restriction endonuclease” (and will be from
this point on in the notes!)
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Type II Restriction Endonuclease (cont.)
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Restriction enzymes may make either staggered cuts
or blunt cuts (flush cuts, straight cuts)
In a blunt cut, the two phosphodiester bonds that are
cut are directly across from each other, so each piece
has double stranded DNA all the way to the end
In a staggered cut, the two phosphodiester bonds
that are cut are offset, so each piece has a short
segment of single-stranded DNA at its end
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Type II Restriction Endonuclease (cont.)
Most restriction sites are molecular palindromes
(palindromic), meaning that the sequence on one
strand reads the same as the sequence on the other
strand, but in the opposite direction
 If the recognition site is palindromic and the enzyme
makes a staggered cut, then the single stranded ends
will be complementary to each other. These are
called sticky ends.
 Sticky ends made with the same enzyme can
hybridize, allowing DNA from more than one source
to be spliced together. The segments are sealed
together with a different enzyme, called DNA ligase.
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Type II Restriction Endonuclease (cont.)
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Example: EcoRI
5’
↓
3’
-N-N-G-A-A-T-T-C-N-N-N-N-C-T-T-A-A-G-N-N3’
↑
5’
5’
3’
-N-N-G
A-A-T-T-C-N-N-N-N-C-T-T-A-A
G-N-N3’
5’
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Definitions
Recombinant DNA: A double stranded DNA molecule
created by splicing DNA from different sources, using
restriction enzymes and DNA ligase
 DNA cloning: using a bacterial species (most often, E.
coli), to replicate recombinant DNA
 Vector: A small double stranded DNA molecule with an
origin of replication for the bacterial host and a system for
selecting recombinant DNA molecules of interest; most
often an engineered bacterial plasmid or bacteriophage.
Example: pUC18
 Recombinant DNA genomic library: A collection of
bacterial colonies with recombinant DNA (for example,
an armadillo library), ideally containing the entire
genome of the species
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Creating a Recombinant Library
(Shotgun approach)
Cut the vector DNA (pUC18) and the genomic DNA
(armadillo DNA, if you want an armadillo library) with
the same restriction enzyme (or combination of enzymes)
 Mix the vector and genomic DNA and ligate using DNA
ligase
 After the ligation step, the mixture will basically contain
three things:
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 Genomic DNA without a vector
 Vector DNA (pUC18) without an insert (no genomic DNA)
 Recombinant DNA consisting of a vector molecule with a
piece of genomic DNA inserted (spliced in)
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Creating a Recombinant Library (continued)
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Use the DNA mixture to transform competent E. coli
cells. After transformation, there will basically be
four kinds of E. coli in the tube:
 E. coli cells that were not transformed (didn’t get any
new DNA)
 E. coli cells that were transformed with “Genomic only”
 E. coli cells that were transformed with “Vector
only/No insert”
 E. coli cells that were transformed with “Recombinant
plasmids/With Insert”
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Creating a Recombinant Library (continued)
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Plate the transformed E. coli onto X-gal (5-bromo-4chloro-3-indolyl-β-D-galactopyranoside) agar plates.
These are selective and differential plates.
 X-gal agar contains ampicillin. If the E. coli cells weren’t
transformed or got genomic DNA only (no vector/no
plasmid), the ampicillin kills them.
 If the transformed E. coli got a vector only/no insert, it forms
a blue colony.
 If the transformed E. coli got a vector with a genomic DNA
insert, it forms a white colony.
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White colonies are further screened to determine which
genes they contain. A total armadillo library might
require screening of several thousand colonies!
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Origin of replication (ori) from one or more
bacterial species
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“Shuttle vector” has origins from two or more
species, allowing cloned genes to be shuttled from
one species to another
An antibiotic resistance gene (e.g. ampR). By
using medium containing the antibiotic
(ampicillin), only cells transformed with the
vector DNA can survive. This selects against
untransformed cells.
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One or more restriction enzyme recognition sites
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“Polylinker” – This is an engineered DNA segment containing
recognition sites for several different enzymes, in tandem.
A way to screen for vectors with inserts vs vectors without
inserts
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Typically, this is a combination of the lac z gene (β-galactosidase
gene) and the lac promoter sequence (required for transcription of
the lac z gene). With no insert, a transformed cell will make βgalactosidase.
The polylinker is engineered to overlap or sit between the lac p
and lac z sequences.
If there is an insert in the vector, it separates or disrupts lac p and
lac z, so that β-galactosidase is not made.
X-gal agar contains a synthetic substrate of β-galactosidase that
turns blue if the enzyme is present (no insert).
If there is an insert present, then β-galactosidase is not made, so the
colonies are white.
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Two approaches
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Screen for expression of the heterologous protein
Use a labelled DNA hybridization probe to search
for colonies with homologous sequences, using
blotting techniques
Blotting techniques
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Southern Blotting: Use a labeled DNA probe to
analyze DNA fragments, separated by agarose gel
electrophoresis and transferred (“blotted”) onto
nitrocellulose or nylon membrane sheets
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Blotting techniques
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Northern Blotting: Use a labeled DNA probe to
analyze RNA molecules, separated by agarose gel
electrophoresis and transferred (“blotted”) onto
nitrocellulose or nylon membrane sheets
Dot Blotting: The unknowns are simply spotted onto
a membrane, then analyzed with a DNA probe
Western Blotting: Not really a DNA technique.
Analyzing proteins on a nylon membrane using a
labeled antibody molecule as a probe
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Blotting techniques
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Northern Blotting: Use a labeled DNA probe to
analyze RNA molecules, separated by agarose gel
electrophoresis and transferred (“blotted”) onto
nitrocellulose or nylon membrane sheets
Dot Blotting: The unknowns are simply spotted onto
a membrane, then analyzed with a DNA probe
Western Blotting: Not really a DNA technique.
Analyzing proteins on a nylon membrane using a
labeled antibody molecule as a probe
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Approaches to obtaining a DNA probe
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Use a homologous sequence from a different (ideally
one that is related) species
Isolate mRNA for the gene of interest (for example,
by using antibodies to immunoprecipitate the
ribosome/nascent protein/mRNA complex). You
then use reverse transcriptase to make a cDNA copy
of the mRNA.
Use DNA chemical synthesis techniques to create
possible homologous sequences to the gene of
interest, and test them as probes.
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After a segment of DNA (for example, from our aardvark library)
has been isolated, it is routinely sequenced.
In four separate tubes, the aardvark DNA is added to a DNA
replication mixture containing nucleotides, DNA polymerase,
and...
A labeled dideoxynucleotide. Each tube gets a different
dideoxynucleotide, either ddA, ddT, ddC, or ddG
When a dideoxynucleotide gets added during DNA replication, it
causes chain termination, which means that replication stops
The labeled fragments from the four mixtures (corresponding to
A, T, C, and G) are separated by size using polyacrylamide gel
electrophoresis)
By arranging the fragments in order of size, the sequence of the
DNA is determined.
In automated sequencers, a single mixture is used, with different
fluorescent labels for each nucleotide, and the fragments are
separated by capillary electrophoresis and analyzed automatically
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A DNA sample can be amplified in a test tube without
the need for cloning, using the Polymerase Chain
Reaction (PCR) technique
The DNA is replicated using a thermostable DNA
polymerase (for example, the Taq polymerase from
Thermus aquaticus) with alternating rounds of heating
and cooling in a device called a thermal cycler
Since DNA polymerase requires a short segment of
DNA to begin replication (a primer), the exact
sequence amplified can be controlled by choosing the
appropriate primer
This technique can be used with a very small starting
sample – for example, the DNA from a single hair
strand at a crime scene
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The use of genomic sequence databases to find
genes and to predict their behavior
Closely connected with
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Genomics, the analysis of the complete genomic
sequence of a species; and
Proteomics, the analysis of all the proteins made by a
species