Nucleic Acids Lecture 23 (11/10/21)
A. Nucleotides
B. Nucleic Acids • Reading: Ch9; 309-313
C. The 4 S’s Ch8; 283-286
1. Size
2. Solubility
3. Shape
a. A-DNA • Problems: Ch8; 13
b. Z-DNA
c. Topology
i. Packaging
ii. Supercoiling
iii. Topoisomerases NEXT
4. Stability
a. Nucleotides • Reading: Ch25; 916-921, 930-940
i. Tautomers
ii. Acid/base
b. Nucleic Acids
i. Chemistry • Problems: Ch25; 2,3,5,6,7,9,10,13,14,
ii. Denaturation 15,16,17
iii. Stability
iv. Nucleases
1. X
D. Structure of the Information 2. X
1. Exceptions to flow 3. Transformation of hosts
2. Structure 4. Selection of transformants
3. Levels of Control a. Selectable marker/gene
b. Distinguish empty plasmids
E. Recombinant DNA: Biochemical Basis of Biotechnology i. Loss of resistance
1. Restriction enzymes, DNA ligase ii. Reporter gene
2. Vectors and Inserts to make recombinant DNA (rDNA) 5. Expression
a. Inserts a. Special vectors
i. cDNA b. Fusion proteins
ii. Genomic i. purification
b. Vectors ii. labeling
6. Site-directed mutagenesis
Recombinant DNA and
Biotechnology
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Recombinant DNA and Biotechnology
Recombinant DNA is DNA made in the laboratory that is
derived from at least two genetic sources.
Recombinant DNA has allowed molecular biology to come
full circle.
FUNCTION
Biochemistry Genetics
PROTEIN GENE
recDNA
Recombinant DNA has one simple goal: MAKE MORE
Recombinant DNA and Biotechnology
Recombinant Proteins
………coronavirus
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Recombinant DNA and Biotechnology
Recombinant DNA is DNA made in the laboratory that is
derived from at least two genetic sources.
• Biochemical Basis of Biotechnology
- Restriction enzymes, DNA ligase
- Vectors and Inserts to make recombinant DNA
(rDNA)
- Transformation of hosts
- Selection of transformants
- Expression
- Site-directed mutagenesis
Recombinant DNA and Biotechnology
Restriction enzymes are used to
cut DNA into fragments, which then
are spliced together in new
combinations.
DNA ligase catalyzes the joining of
DNA fragments.
Others you will see how they are
used when we go through the
processes.
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Recombinant DNA and Biotechnology
Restriction Sites
Restriction enzymes recognize palindromic DNA sequences:
5ʼ…….GAATTC……3ʼ
3ʼ…….CTTAAG……5ʼ
Some make straight cuts, others make staggered cuts, resulting in
overhangs or sticky ends.
EXAMPLES:
Recombinant DNA and Biotechnology
Restriction Sites
Restriction enzymes recognize palindromic DNA sequences:
5ʼ…….GAATTC……3ʼ
5ʼ…….G-3ʼ 5ʼ-AATTC……3ʼ
3ʼ…….CTTAAG……5ʼ
3ʼ…….CTTAA-5ʼ 3ʼ-G……5ʼ
Some make straight cuts, others make staggered cuts, resulting in
overhangs or sticky ends.
EXAMPLES:
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Recombinant DNA and Biotechnology
Restriction Endonucleases
EcoRI
Endonuclease
EcoRI endonuclease
PDBid 1ERI
EcoRV
Endonuclease
EcoRV endonuclease
PDBid 4RVE
Restriction Endonucleases Cleave at
Specific Recognition Sites
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Restriction Endonucleases Key to
Making rDNA Molecules
DNA Ligase will “seal” the “nick” by
making the covalent
phosphodiester bond
DNA Ligase: Also Key to Making rDNA
Molecules Enzyme from E. coli
NAD+
AMP NMN+
Adenine–Ribose–℗–℗–Ribose–Nicotinamide
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DNA Ligase Reaction
Enzyme from E. coli
DNA Ligase
NAD+
AMP NMN+
Adenine–Ribose–℗–℗–Ribose–Nicotinamide
Restriction Endonucleases & DNA
Ligase: Key to Making rDNA Molecules
Process Diagram:
Recombinant DNA Construction
Recombinant DNA
molecule
Herbert Boyer & Stanley Cohenr
Where does this Foreign DNA come from and how is it purified?
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Recombinant DNA and Biotechnology
Vectors and Inserts DNA fragments used for molecular cloning come from two sources:
• Genomic DNA
• cDNA (Copy DNA or complementary DNA)From reverse transcription of mRNA
Represents an mRNA from
A genomic clone a given cell/tissue. cDNA
contains the is produced by making a
DNA copy of the mRNA
gene(s) as a
fragment of the population using the RNA-
genome of an directed DNA polymerase
called, reverse
organism.
transcriptase.
The DNA is cut into
fragments by A cDNA library is a
“snapshot” of the
restriction
enzymes, and transcription pattern of the
each fragment is cell.
inserted into a cDNA clones are used to
vector. provide the ORF for
expressing the protein
A genomic clone A cDNA clone
Recombinant DNA and Biotechnology
Vectors and Inserts DNA fragments used for molecular cloning come from two sources:
• Genomic DNA
• cDNA (Copy DNA or complementary DNA)From reverse transcription of mRNA
Represents an mRNA from
A genomic clone a given cell/tissue. cDNA
contains the is produced by making a
DNA copy of the mRNA
gene(s) as a
fragment of the population using the RNA-
genome of an directed DNA polymerase
called, reverse
organism.
transcriptase.
The DNA is cut into
fragments by A cDNA library is a
“snapshot” of the
restriction
enzymes, and transcription pattern of the
each fragment is cell.
inserted into a cDNA clones are used to
vector. provide the ORF for
expressing the protein
Where does this
Foreign DNA
come from and
A genomic clone A cDNA clone how is it purified?
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Recombinant DNA and Biotechnology
Vectors and Inserts: cDNA Cloning
Recombinant DNA and Biotechnology
Vectors and Inserts: cDNA Cloning
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How do you find your DNA of interest?
Restriction Digest Electrophoretogram
Process Diagram: Southern Blotting
Using
polynucleotide
kinase and g-
32P-ATP
Recombinant DNA and Biotechnology
• Biochemical Basis of Biotechnology
- Restriction enzymes, DNA ligase
- Vectors and Inserts to make recombinant DNA
(rDNA)
- Transformation of hosts
- Selection of transformants
- Expression
- Site-directed mutagenesis
All vectors have 3 things:
1. Autonomous replication ability
2. Selection for hosts that contain vector
3. Site for insertion of rDNA
Viruses as vectors (bacteriophage or
retroviruses).
• Viruses can be altered to attenuate some
detrimental genes in the virus (e.g., those
that kill the host).
• Viruses can be altered to carry recombinant
DNA into cells
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Recombinant DNA and Biotechnology
• Biochemical Basis of Biotechnology
- Restriction enzymes, DNA ligase
- Vectors and Inserts to make recombinant DNA
Inserting the recombinant DNA into a cell:
(rDNA) • Cells may be treated with chemicals to
- Transformation of hosts make plasma membranes more
permeable—DNA diffuses into cells.
- Selection of transformants • Electroporation—a short electric shock
- Expression
Transformation: Recombinant DNA is cloned creates temporary pores in membranes,
by inserting it into host cells (transfection if and DNA shoots to the + end and can enter
host-cells
Site-directed mutagenesis cells.
are from an animal). Second key
discovery in biotechnology.
Usually only a few cells are transformed (1 cell in
10,000). Reason for the need for a selectable
marker.
The first host cells used were bacteria, especially x1 x 10,000
E. coli.
Yeasts (Saccharomyces) are commonly used as
eukaryotic hosts.
Recombinant DNA and Biotechnology
• Biochemical Basis of Biotechnology
- Restriction enzymes, DNA ligase
- Vectors and Inserts to make recombinant DNA
(rDNA)
- Transformation of hosts
- Selection of transformants
• Use-ofExpression
antibiotic resistance gene (e.g., ampicilin
- Site-directed
resistance) on a plasmid mutagenesis
• For viral vectors, use of “infected” phenotype.
• Use of “selectable markers” to detect either
insertion into the vector or incorporation into the
host. Some of these are a type of reporter
gene—a gene whose expression is easily x1 x 10,000
observed.
• Many plasmids contain the lacZ gene with a
multiple cloning site within its sequence. lacZ
codes for an enzyme that can convert the
substrate X-Gal into a bright blue product
Plasmid Cloning Video
How can you distinguish between
recDNA vectors and ”empty” vectors?
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Recombinant DNA and Biotechnology
DNA Cloning
Introduce DNA into Organism Use of pBR322 to clone foreign DNA in E. coli and identify
cells containing it.
Antibiotic Selection
• Antibiotics, such as penicillin and ampicillin, kill
bacteria.
• Plasmids can carry genes that give a host
bacterium a resistance against antibiotics.
• Allows growth (selection) of bacteria that have
taken up the plasmid
OH
O
O
N O
S N
H H
H N
H
Identification of Empty Plasmids
Ampicillin
Selection using Reporter Gene
Insert ORF
Plac of interest
• Origin of replication
• Antibiotic resistance gene
• Multiple cloning site
• Promoter for transcription,
translation (host specific)
Recombinant DNA Clone
Ori
(–) Insert – Makes b-Galactosidase
(+) Insert – Doesn’t
make b-Galactosidase
N/A
N/A
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Recombinant DNA and Biotechnology
• Biochemical Basis of Biotechnology
- Restriction enzymes, DNA ligase
- Vectors and Inserts to make recombinant DNA
(rDNA)
- Transformation of hosts
- Selection of transformants
- Expression
- Site-directed mutagenesis
Expression vectors include sequences needed for
expression of a transgene in a host cell.
• For prokaryote host, which are preferred for
making large amounts: A bacterial promoter,
ribosome binding site, and a transcription
termination signal, must all be included.
• For eukaryote (mostly yeast): The
eukaryotic promoter/enhancer and
terminator (poly-A addition signal/site) must
be included.
These can also be:
• Inducible promoters which respond to a specific signal
• Tissue-specific promoters expressed only in certain tissues at
certain times
• Signal sequences—e.g., a signal to secrete the product to the
extracellular medium
Recombinant DNA and Biotechnology
Typical Expression Vector
Insert ORF
of interest
Can you use such an
expression system
to help in
purification?
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Recombinant DNA and Biotechnology
Purification of Recombinant Proteins
• Purification of natural proteins is difficult.
• Recombinant proteins can be tagged for purification.
• The tag binds to the affinity resin, binding the protein
of interest to a purification column.
TABLE 9-3 Commonly Used Protein Tags
Tag protein/peptide Molecular mass (kDa) Immobilized ligand
Protein A 59 Fc portion of IgG
(His)6 0.8 Ni2+
Glutathione-S-transferase
26 Glutathione
(GST)
Maltose-binding protein 41 Maltose
p-Aminophenyl-β-D-
β-Galactosidase 116
thiogalactoside (TPEG)
Chitin-binding domain 5.7 Chitin
Recall:
Affinity Chromatography Basis =
Biotechnology (recombinant DNA technology) has function
revolutionized protein purification.
At the level of the DNA sequence, the DNA sequence
encoding such binding proteins or ”tags” can be “fused”
to the sequence encoding YFP. In this way, a chimeric
protein is produced that has the binding function, which
allows the use of affinity chromatography.
Common “tags” are: Column beads have attached:
Maltose-binding protein Maltose
Chitin-binding protein Chitin
Glutathione-S-transferase (GST) Glutathione (g-Glu-Cys-Gly)
His-His-His-His-His-His Ni-chelate
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