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Chapter 5 Notes

Biochemistry 461

Fall 2010

CHAPTER 5, EXPLORING GENES:

LECTURE TOPICS
1) RESTRICTION ENZYMES
2) GEL ELECTROPHORESIS OF DNA
3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS
4) POLYMERASE CHAIN REACTION (PCR)
5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING
6) DNA CLONING VECTORS
7) GENE LIBRARIES: MAKING AND SCREENING THEM
8) CHROMOSOME MAPPING
9) EXPRESSION OF CLONED GENES
10) ENGINEERING NOVEL PROTEINS

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Recombinant DNA technology (started in late 1970's)

!

An incredibly powerful set of tool for gene manipulation.

Methods associated with this "technology" make genetic engineering a

reality.

DNA (genes), RNA, and protein structure and function can be altered by
design for beneficial (or detrimental -biological warfare/terrorism?) results.

KEY TOOLS and METHODS OF GENE EXPLORATION

!

ENZYMES to cut, join and replicate DNA in test tubes (in vitro)
a) restriction enzymes are DNA cutters
b) DNA ligases are DNA joiners
c) DNA replication requires DNA polymerases

GEL ELECTROPHORESIS to separate and isolate specific DNAs

BLOTTING METHODS based on hybridization (BASE-PAIRING) of

complementary DNA and/or RNA

SOLID PHASE methods to sequence and synthesize DNA

POLYMERASE CHAIN REACTION for gene detection and amplification.

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RESTRICTION ENZYMES (ENDONUCLEASES)

DNA scissors - hundreds of restriction enzymes are known

Recognition sequences - different lengths (often 4-8bp), palindromic (2 -fold

rotational axis of symmetry), specific cleavage sites (Fig. 6.1)

They can leave overhanging ends or blunt ends

Named (ex: HindIII) for source bacterial

strain:
H = Haemophilus in = influenzae
d = strain d III = third one identified

Number of cuts in a specific DNA ranges from

few (if long recognition site) to many (short or ambiguous recognition site)

patterns of fragments are diagnostic of a given DNA species and physical maps
of whole chromosomes can be made. (Fig.6.-2)

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GEL ELECTROPHORESIS OF DNA

Agarose gels separate DNA restriction fragments

Visualize DNA by staining or autoradiography

even differences of one base pair can be detected on gels.

Hybridization - Base-pairing of DNA-DNA, DNA-RNA: Complementary singlestranded DNA and RNA molecules form base-paired structures even if only 2 or
3 bases can pair - like at ends of restriction fragments!

[IMPORTANT: Hybridization (DNA-DNA, DNA-RNA) is almost always used in

one or more ways to to detect particular DNA or RNA sequences and to
construct new combinations of DNA fragments.]
NUCLEIC ACID BLOTTING AND HYBRIDIZATION: DNA bands (and patterns) on gels

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can be transferred to nitrocellulose filters (Southern blotting) and identified by
hybridization with a specific gene probe. (Fig.6.3)

Southern (DNA), Northern (RNA), and Western (protein) blotting methods are
all powerful probes of gene function.

Rest
riction
fragment length polymorphism (RFLP) gel analysis is a powerful diagnostic
tool (ex. sickle-cell anemia genetic typing)
MstI RFLP for Sickle-Cell Mutation Detection [Fig 7-52]
Normal

Sickle cell

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LANDMARK DNA SEQUENCES COMPLETED
5386 bases
155,844 bases
1.8 million bases
3 million bases
bases

tRNA - (1964) (
, complicated method)
NX174 DNA (1977)
tobacco chloroplast DNA (1986)
H. influenzae (1995)
E. coli (1997)
human (2000!!)

DNA SEQUENCING: ALL METHODS REQUIRE THE FOLLOWING*

reactions specific for each base

controlled random reactions

equimolar collection of reaction products (same frequency of

stopping each time the same base occurs)

DNA SEQUENCING BY CONTROLLED RANDOM CHAIN TERMINATION OF DNA

SYNTHESIS METHOD (SANGER DIDEOXY METHOD)
(Fig.6.4)

Use a template-primer complex, a DNA polymerase, dNTPs, and one 2'-3'

dideoxynucleoside triphosphate (one for each base) in each of four reactions.

DNA synthesis occurs (specific for each base) until a dideoxy nucleoside
phosphate is inserted into the nascent DNA - then the reaction stops! (no free
3'-OH group to attack the incoming dNTP substrate).

Reaction is carried out under conditions (adjust ratio of dNTP: ddNTP) to give
equal representation and distribution of products.

Run reaction products on gel. 500-600 bases can be easily read on one gel.

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Strategy for Chain termination
DNA sequencing: (Fig.6.4)

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Current methods for DNA/RNA sequence determination:

Use fluorescent-labelled nucleotides (makes each base reaction mix flouresce

a different color); mix all 4 reaction products, and detect all with automated
fluorometers to detect DNA reaction products and analyze by computer
immediately.(Fig. 6.5) [Can sequence whole genomes (ex. Fig.6.6 - bacterial)

Can also sequence DNA by detecting hybridization signals on arrays of short

DNA sequences bound to microchips.

RNA SEQUENCING - RNA can be sequenced directly, but now it is done by

dideoxy method, using reverse transcriptase with a synthetic DNA primer.

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CHEMICAL SYNTHESIS OF DNA (SOLID PHASE, AUTOMATED METHODS)

oligodeoxynucleotide chain (short DNA, parts of genes for probes and primers)

aid DNA/RNA sequencing, cloning, and gene probing by hybridization

easy to make DNA 100 nucleotides long (18-20 used most often)

Chemically synthesized DNAs are key to protein engineering by site-directed

mutagenesis.

Start with blocked nucleotide linked

to a solid support (glass bead).

protected nucleotides added

stepwise (but 3' to 5' - reverse of
DNA polymerase)

Stepwise linking of activated

monomers (deoxyribonucleoside 3'phosphoramidites) that are blocked
at 5'-P (and have protected amino
groups)

Chemical synthesis of DNA: (Fig.6.7)

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POLYMERASE CHAIN REACTION (PCR) Discovered in 1984

[K.B. Mullis,Scientific American,1990, 262:56-65)]

Incredibly powerful method to amplify (synthesize) DNA starting with as little as

one copy of a DNA molecule. (Figs.6.8 and 6.9)

PCR CONCEPT (Fig.6.8): Two oligonucleotides

spanning a gene of interest and on opposite
strands are used to prime multiple cycles of DNA
synthesis catalyzed by a heat stable DNA
polymerase (ex., Taq polymerase from a
thermophilic bacterium).

Ex:PCR starting with one of 2 strands of DNA.

**See Fig 6.9 for

drawing of three
cycles of PCR

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PCR RESULT: In 25-40 replication cycles, enough DNA is synthesized to

clone or to sequencedirectly.

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PCR USES: Forensics, diagnosis of genetic defects in utero, sequencing DNA

from fossils, detecting small amounts of bacteria (ex; anthrax), etc.

FORENSICS EXAMPLE: Bloodstain analysis of victim (V) and clothing of

defendant (D - apparently guilty!). (Fig.6.10)

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CONSTRUCTION, CLONING AND EXPRESSION OF DNA:

Novel combinations of genes can be constructed, cloned, amplified and
expressed in foreign environments.
KEY STEPS/PROCEDURES/TOOLS [Fig.6.11, 4th Ed.]

Construct recombinant molecule (link DNA insert with a vector).

Amplify and clone DNA - Introduce DNA into host cells as naked DNA or as
DNA incorporated into virus particle. DNA must be replicated autonomously in a
host cell.

Selection by antibiotic resistance, gene probing, antibody reaction

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CONSTRUCT RECOMBINANT MOLECULE - link a DNA insert with a vector.
(Figs.6.11,12)
CUT AND JOIN DIFFERENT DNA MOLECULES: Restriction enzymes and DNA
ligase.

Restriction enzymes that give cohesive ends (short complementary ends

that can base pair) cut unique cloning sites in vectors.[Ex: Eco RI (Fig.6.11)]

The vector and insert are covalently linked with DNA ligase.

Restriction enzymes that give blunt ends need linkers added by T4

DNA ligase to get cohesive ends for cloning. [Ex: Eco RI linker (Fig.6.12)]

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Amplify and clone DNA: Introduce DNA into host cells as naked DNA [See
illustration page 13] or as DNA incorporated into virus particle. DNA must be
replicated autonomously in a host cell.
Selection of recombinant DNA-containing host cells - antibiotic resistance, gene
probing, antibody reactions, etc.

SOME CLONING VECTORS (autonomously replicating)

Plasmids: Insertional inactivation of antibiotic resistance often used. Use for
small DNAs.
Lambda phages (Larger DNA pieces tha plasmids. Especially useful for
libraries of cDNA or eucaryotic genomic DNA)
YACs and BACs (yeast and bacterial artificial chromosomes) - for long DNA,
especially big pieces of chromosomes.

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CLONING VECTORS

Plasmids (accessory chromosomes which

replicate autonomously) are excellent vectors for
cloning DNA of 2-6 kb in E. coli. Antibiotic
resistances are used for selection of
recombinant plasmids. Insertional inactivation
signals the presence of a DNA insert in an
antibiotic resistance gene. (See page 15 and
Fig.6.13)

Special lambda (8) phages (they work a lot like

T2 bateriophage) used to clone large pieces (10-20kb) of DNA between each
end of lambda DNA. Especially suitable for cloning of libraries of cDNA or
eucaryotic genomic DNA. (Fig.6.14,15)

lambda (8) phage

lifecycle

Cloning DNA
between each end of
lambda (8) DNA

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Yeast artificial chromosomes (YACs) - Can clone large pieces of DNA

(100,000 to a million base pairs). (Fig.6.21)

GENE LIBRARIES: What are they??

Genomic library. Made from all restriction fragments of a cell's DNA. A collection of
cloned sequences which represents a whole genome.

Genomic library vectors: Bacteriophage lambda, YACs and BACs

cDNA library. Made from mix of all of a cells mRNAs. Use reverse transcriptase to get
DNA. A cDNA library is a mix of DNAs complementary to ALL genes that
are being expressed as mRNAs.

CDNA library vectors: lambda phage, plasmids

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Constructing a genomic library in bacteriophage 8. Made from all restriction
fragments of a cell's DNA. A collection of cloned DNA sequences which represents
a whole genome.

Constructing a cDNA library from a cells mRNAs. Use reverse transcriptase to get
DNA. A cDNA library is a mix of DNAs that represents all of the cells mRNAs.

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SCREENING GENE LIBRARIES: searching for a needle in phagestack!

!
!
!
!
!
!
!

screen 500,000 clones for a specific sequence in a genomic library

Easier for abundant mRNA molecules in a cDNA library.
Hybridization screening with gene probe
Synthetic DNA probes (predict sequence by reverse translation of protein
sequence to a DNA sequence)
Immunochemical (antibody) screening of an expression library
Chromosome walking to connect long pieces fo chromosomes
Can map whole chromosomes - use lambda or YACs for cloning

Screening for a specific

gene with a radioactive
gene probe. (Or antibodies
or fluorescence)

Screening a cDNA library.

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REVERSE TRANSLATION: Make a gene probe from a known protein sequence.

!
!
!

Predict DNA base sequence from amino acid sequence, using Genetic Code
Synthesize DNA probes from the predicted gene sequences
This example (Fig.6.20) requires a mix of 256 different oligonucleotides to
guarantee a perfect match. (These mixtures really work!!)

CHROMOSOME WALKING: Use to map/explore long regions of chromosomes by

iterative hybridization, subcloning, and rescreening.

DNA fragments near ends of one clone are used to identify longer clones which
contain their sequence and adjacent sequences extending past the original
DNAs ends. (Fig.6.22)

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EXPRESSION OF CLONED GENES
DNA vector delivery to cells.

!
!
!
!
!
!

calcium phosphate precipitated DNA

microinjection
virus vectors(SV40), vaccinia, retroviruses (ex: Maloney
murine leukemia virus)
GENE GUN" (microprojectiles coated with DNA)
liposomes
electroporation

Expression vectors:

!
!
!

Designed to give efficient transcription and translation by cloning a gene

near a strong promoter.
The gene must be cloned in the correct reading frame with a properly
spaced ribosome binding site.
Immunochemical screening will identify clones, if an antibody to the
cloned protein is available.

SOME EXAMPLES

Proinsulin cDNA was cloned in a plasmid and the proinsulin was made by E.
coli cells. This is a standard method to express cloded genes as proteins, and is
the basis for much of the biotechnology industry. (Fig.6.23)

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Genetically engineered giant mice result from injection of somatotropin gene

into male pronucleus of a fertilized mouse egg. Cd++ controls expression of this
gene by its placement under control of the metallothionein gene. [Fig. 6-32]

Plant Genetic Engineering. Genes are cloned into the Ti-plasmid of

Agrobacterium tumefaciens. Part of this plasmid (the T-DNA) is incorporated
into plant chromosomes after Agrobacterium infection. DNA which is cloned
between the right and left ends of the T-DNA can be expressed by "transformed"
plant cells after integration in a chromosome. (Figs.6.33,34)

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Ti-plasmid of Agrobacterium tumefaciens

Gene disruption/replacement. Genes can be inactivated (knockout mutant) or

replaced by a modified or completely new gene by homologous recombination

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ENGINEERING NOVEL PROTEINS:

Modify DNA coding information to get protein with different amino acid sequence.

These are really novel combinations which would not occur in nature.

Solid phase synthesis of whole genes of any type is now possible.

Site-specific mutagenesis - Hybridize synthetic oligonucleotide with a mismatched

base (Fig.6.36)

Cassette Mutagenesis: (Fig.6.37)

Use restriction fragments to

combine parts of genes coding for
different domains of different
proteins.

introduce a DNA fragment with

one or more changes from normal
gene.

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SUMMARY: Recombinant DNA technology Roadmap

Can start with a DNA sequence and ultimately isolate an unknown protein. Also
can start with a known protein and isolate its gene. (Fig. 6-38)

CURRENT AND FUTURE APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY

Chromosome mapping and sequencing

Discovery of molecular bases of development, evolutionary relationships

New proteins with new functions (or old proteins with new functions!)

human hormone synthesis in bacteria

antiviral agents

AIDS vaccine development

new pharmacological agents (proteins, RNA, DNA)

antisense RNA therapy

medical diagnostic reagents (gene probes) for detection of genetic diseases,

infections and cancers

gene delivery with disarmed viruses to alleviate diseases caused by known gene
defects.

agricultural revolution with animals having altered traits, more nutritious plants,
heat/drought resistant crops, etc.

forensics - molecular detectives