Recombinant DNA Technology
•restriction enzymes
•making recombinant DNA molecules
•expression vectors
•applications of recombinant DNA technology
Forensic DNA Profiling
•short tandem repeats
•DNA fingerprints
References: p. 514-517; 519-523; 525-526; 543-544
Bacterial restriction endonucleases
A restriction endonuclease is an enzyme that recognizes a
specific nucleotide sequence in DNA (a restriction site)
and makes a cut at that site.
•restriction sites are palindrome sequences
•restriction digestion of DNA results in molecules with
complementary single-strand tails (sticky ends)
Restriction endonucleases
Creating recombinant
DNA molecules
Putting together
pieces of DNA
form different origins
Use of the pUC19 expression vector
•ampR is the ampicillin resistance gene
•the polylinker region inside the lacZ+ gene is a cluster of
restriction sites; any of which maybe chosen to insert a
gene of interest (and this would disrupt the lacZ+ gene)
•any gene inserted in the polylinker region will be under the
control of the T7 virus promoter (which is recognized by
the T7 RNA polymerase) and the lac operator (lacO, the
binding site of the lac operon repressor, which is coded
by lacI)
•the host bacterium for this plasmid is a cell that has been
engineered to carry the viral T7 RNA polymerase gene
under the control of the lac operon promoter and operator
in its genome
Use of the pUC19 expression vector
IPTG is a lactose analog and can therefore induce the
expression of both the T7 RNA polymerase gene in the
host genome and of any gene in the plasmid polylinker
(by binding the lac operon repressor and inactivating it).
Cloning vector pUC19
Creating recombinant
DNA molecules with
a cloning vector
The use of antibiotics for selection
Antibiotic selection: if the nutrient growth medium also
contains an antibiotic, the only bacteria that will be able to
multiply and form colonies will be those that carry the correct
antibiotic resistance gene. Example: ampR in pUC19.
The pUC18 polylinker region inside the lacZ+ gene
pUC18 is very similar to pUC19.
The Xgal blue-white assay to identify E. coli colonies
that contain cloned DNA fragments
To screen for cells that have picked up a plasmid with a
cloned DNA fragment following transfection, cells are
grown on agar medium containing X-gal, the antibiotic
ampicillin, and IPTG.
•any bacterium that takes up a plasmid will become
resistant to ampicillin and will grow to form a colony
•recombinant plasmids (those with a foreign DNA fragment
inserted in their polylinker regions) will have a disrupted
lacZ+ gene and will not express b-galactosidase
•plasmid vectors that did not integrate a foreign DNA
fragment in their polylinker regions will have an intact
lacZ+ gene and will express b-galactosidase
The Xgal blue-white assay to identify E. coli colonies
that contain cloned DNA fragments
The enzyme b-galactosidase
cleaves X-gal into galactose
and 5-bromo-4-chloro-3hydroxyindole, which
is then oxidized to an
insoluble blue dye.
The Xgal blue-white assay to identify E. coli colonies
that contain cloned DNA fragments
•Blue colonies (X-gal positive) carry plasmid vectors without
cloned foreign DNA.
•White colonies (no Xgal reaction) carry plasmids with
cloned foreign DNA.
The Agrobacterium Ti plasmid can be used to transfer
genes into plants
TL and TR: flanking sequences that are required for the
transfer of the DNA segment from bacteria to the plant cell.
The Agrobacterium Ti plasmid can be used to transfer
genes into plants
Generating transgenic crops with glyphosate resistance
Glyphosate is a herbicide that kills plants by inhibiting EPSP
synthase, a chloroplast enzyme that is essential for amino
acid biosynthesis.
•EPSP synthase can be expressed in larger than normal
quantities by plants that carry the EPSP gene under the
control of the cauliflower mosaic virus (CMV) promoter
in a Ti plasmid vector
•plant crops that carry this integrated plasmid are resistant to
glyphosate concentrations that would kill normal plants
(a strong
promoter)
Callus: a cluster of undifferentiated cells
that results from the proliferation of a
Ti-transformed cell in culture. A plant
cell callus can regenerate a whole plant.
Gene targeting in mouse ES cells
Mutant organisms can be created to study the effect of mutant
alleles in vivo. Knockout mice can be obtained after
homologous recombination of a cloned mutant allele with
the cellular gene in embryonic stem (ES) cells.
Generation of knockout ES cells:
•the cloned gene of interest (target) is disrupted with a
segment of DNA containing the neomycin resistance gene
(neo+)
•the disrupted gene is placed in a vector that also contains the
thymidine kinase (tk+) gene from the herpes simplex virus
(thymidine kinase can phosphorylate the thymidine analog
glanciclovir, turning it into a toxic inhibitor of DNA
replication and killing the cell)
•the vector is introduced into mouse ES cells
Gene targeting in mouse ES cells
Two thing may happen in the ES cells:
•the homologous recombination of the disrupted target gene
with the cellular gene (knockout), replacing a copy of the
normal cellular target with the disrupted version (these
cells will be able to grow in medium containing both
neomycin and ganciclovir because the tk+ gene will not be
integrated)
•the random integration of the entire vector into the cellular
genome without knocking out the normal cellular target
(these cells will not be able to grow in medium containing
ganciclovir because the tk+ gene will be integrated and
expressed, resulting in toxicity)
Creating knockout mice
Creating knockout mice
Once created, the knockout ES cells are injected into a mouse
embryos.
•knockout ES cells from a black mouse are injected into the
embryo from a white mouse
•the hybrid embryo is implanted in a foster mother and
develops into a chimeric mouse (which may have black
mouse germ cells)
•the chimera mice are mated to obtain homozygous knockout
mice (black)
Gene therapy
Components of the Moloney Murine Leukemia Virus
(MLV) genome:
•the Y gene, necessary for encapsulation
•structural genes of the virus
- gag: coat proteins
- pol: reverse transcriptase
- env: envelope glycoproteins
•5’ and 3’ long terminal repeats (LTRs)
In the SAX virus, all the structural genes of MLV have been
replaced:
•neor: neomycin (antibiotic) resistance
•SV40: simian virus promoter/enhancer
•hADA: Human adenosine deaminase (ADA) gene
Gene therapy
The SAX virus can be encapsulated but will not reproduce.
It can be used as a gene therapy vector to correct severe
combined immunodeficiency (SCID), a hereditary disease
caused by a defect in the hADA gene.
Gene therapy
Examination of the human genome
Forensic DNA profiling
Forensic DNA profiling is the use of genetic markers to
distinguish individuals and answer questions of interest to
the legal system.
•the markers used are polymorphic loci that contain multiple
repeats of a small unit (a repetitive sequence)
•the number of repeats of the unit sequence in each
polymorphic marker locus vary widely among individuals,
and their electrophoretic profile yields DNA fingerprints
that are unique for each individual
Short tandem repeats
(STRs)
STRs are clusters of tandem repeats of 2-9 nucleotide
sequences.
•the lengths of the clusters depend on the number of sequence
repeats in each allele
•because the small sequence sizes, DNA analysis can be
done by using a method that is quick and requires very
little tissue sample: the Polymerase Chain Reaction (PCR)
•the results yield a DNA fingerprint
•STR loci are also known as PCR loci
Short tandem repeats
The D7S280 STR locus
1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt
61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa
121 ctaacgatag atagatagat agatagatag atagatagat agatagatag atagacagat
181 tgatagtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg
241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc
301 cctctgagtt tttgatacct cagattttaa ggcc
D7S280 is one of the 13 core CODIS STR genetic loci.
This DNA is found on human chromosome 7.
In this particular example, the chromosome has 15 repeats of
the GATA sequence. The number of repeats may be different
in the other chromosome or in another individual.
The FBI’s 13 core STR loci
The amelogenin test
Amelogenin is a protein found in the developing tooth
enamel.
•the gene (AMEL) is present in both the X and the Y
chromosomes, but the X-linked version is shorter
•it can be used for sex determination of human DNA samples
of unknown origin through PCR of intron 1 of the gene:
- PCR of AMEL-X gives rise to a 106 bp product
- PCR of AMEL-Y gives rise to a 112 bp product
DNA profile using several STRs
DNA fingerprints from the victim, an tissue sample of
unknown origin found at the crime scene, and three suspects:
Examples of DNA evidence in forensic identification
Identification of September 11, 2001 victims:
•there were 2603 victims in the attacks to the World Trade
Center, but only 293 whole bodies were recovered
•~20,000 pieces of bone and tissue were found among the 1.6
million tons of debris (some pieces as small as a fingertip)
•laboratories collected personal items from the victims’
homes (~7000 razor blades, combs, toothbrushes, etc.)
•the method of choice for DNA analysis was STR
Examples of DNA evidence in forensic identification
The people’s STR evidence in the 1995 O. J. Simpson
murder trial:
•7 PCR loci in O. J. Simpson’s blood, found at the foyer of
Nicole Brown’s apartment and leading out of the crime
scene to Bundy Drive
•7 PCR loci in Nicole Brown’s blood, found in a pair of
socks in Simpson’s bedroom
•2 PCR loci in Ronald Goldman’s blood, found on a pair of
bloody gloves at Simpson’s home