Recombinant DNA Technology•restriction enzymes•making recombinant DNA molecules•expression vectors•applications of recombinant DNA technologyForensic 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 aspecific 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 recombinantDNA molecules

Putting togetherpieces 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 agene of interest (and this would disrupt the lacZ+ gene)

•any gene inserted in the polylinker region will be under thecontrol of the T7 virus promoter (which is recognized bythe T7 RNA polymerase) and the lac operator (lacO, thebinding site of the lac operon repressor, which is codedby lacI)

•the host bacterium for this plasmid is a cell that has beenengineered to carry the viral T7 RNA polymerase geneunder the control of the lac operon promoter and operatorin its genome

IPTG is a lactose analog and can therefore induce theexpression of both the T7 RNA polymerase gene in thehost genome and of any gene in the plasmid polylinker(by binding the lac operon repressor and inactivating it).

Use of the pUC19 expression vector

Cloning vector pUC19

Creating recombinantDNA molecules with

a cloning vector

The use of antibiotics for selection

Antibiotic selection: if the nutrient growth medium alsocontains an antibiotic, the only bacteria that will be able to

multiply and form colonies will be those that carry the correctantibiotic 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 coloniesthat contain cloned DNA fragments

To screen for cells that have picked up a plasmid with acloned DNA fragment following transfection, cells aregrown on agar medium containing X-gal, the antibioticampicillin, and IPTG.

•any bacterium that takes up a plasmid will becomeresistant 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 disruptedlacZ+ gene and will not express -galactosidase

•plasmid vectors that did not integrate a foreign DNAfragment in their polylinker regions will have an intactlacZ+ gene and will express -galactosidase

The Xgal blue-white assay to identify E. coli coloniesthat contain cloned DNA fragments

The enzyme -galactosidasecleaves X-gal into galactoseand 5-bromo-4-chloro-3-hydroxyindole, whichis then oxidized to aninsoluble blue dye.

The Xgal blue-white assay to identify E. coli coloniesthat contain cloned DNA fragments

•Blue colonies (X-gal positive) carry plasmid vectors withoutcloned foreign DNA.

•White colonies (no Xgal reaction) carry plasmids withcloned foreign DNA.

The Agrobacterium Ti plasmid can be used to transfergenes into plants

TL and TR: flanking sequences that are required for thetransfer of the DNA segment from bacteria to the plant cell.

The Agrobacterium Ti plasmid can be used to transfergenes into plants

Generating transgenic crops with glyphosate resistance

Glyphosate is a herbicide that kills plants by inhibiting EPSPsynthase, a chloroplast enzyme that is essential for aminoacid biosynthesis.

•EPSP synthase can be expressed in larger than normalquantities by plants that carry the EPSP gene under thecontrol of the cauliflower mosaic virus (CMV) promoterin a Ti plasmid vector

•plant crops that carry this integrated plasmid are resistant toglyphosate concentrations that would kill normal plants

(a strongpromoter)

Callus: a cluster of undifferentiated cellsthat results from the proliferation of aTi-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 mutantalleles in vivo. Knockout mice can be obtained afterhomologous recombination of a cloned mutant allele withthe 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 thethymidine kinase (tk+) gene from the herpes simplex virus(thymidine kinase can phosphorylate the thymidine analogglanciclovir, turning it into a toxic inhibitor of DNAreplication 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 thenormal cellular target with the disrupted version (thesecells will be able to grow in medium containing bothneomycin and ganciclovir because the tk+ gene will not beintegrated)

•the random integration of the entire vector into the cellulargenome without knocking out the normal cellular target(these cells will not be able to grow in medium containingganciclovir because the tk+ gene will be integrated andexpressed, resulting in toxicity)

Creating knockout mice

Creating knockout mice

Once created, the knockout ES cells are injected into a mouseembryos.

•knockout ES cells from a black mouse are injected into theembryo from a white mouse

•the hybrid embryo is implanted in a foster mother anddevelops into a chimeric mouse (which may have black mouse germ cells)

•the chimera mice are mated to obtain homozygous knockoutmice (black)

Gene therapy

Components of the Moloney Murine Leukemia Virus(MLV) genome:

•the 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 beenreplaced:

•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 diseasecaused 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 todistinguish individuals and answer questions of interest tothe legal system.

•the markers used are polymorphic loci that contain multiplerepeats of a small unit (a repetitive sequence)

•the number of repeats of the unit sequence in eachpolymorphic marker locus vary widely among individuals,and their electrophoretic profile yields DNA fingerprintsthat are unique for each individual

Short tandem repeats

STRs are clusters of tandem repeats of 2-9 nucleotidesequences.

•the lengths of the clusters depend on the number of sequencerepeats in each allele

•because the small sequence sizes, DNA analysis can bedone by using a method that is quick and requires verylittle tissue sample: the Polymerase Chain Reaction (PCR)

•the results yield a DNA fingerprint•STR loci are also known as PCR loci

(STRs)

1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt 61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa121 ctaacgatag atagatagat agatagatag atagatagat agatagatag atagacagat181 tgatagtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc301 cctctgagtt tttgatacct cagattttaa ggcc

Short tandem repeats

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 ofthe GATA sequence. The number of repeats may be different

in the other chromosome or in another individual.

The D7S280 STR locus

The FBI’s 13 core STR loci

The amelogenin test

Amelogenin is a protein found in the developing toothenamel.

•the gene (AMEL) is present in both the X and the Ychromosomes, but the X-linked version is shorter

•it can be used for sex determination of human DNA samplesof 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 ofunknown origin found at the crime scene, and three suspects:

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

Examples of DNA evidence in forensic identification

The people’s STR evidence in the 1995 O. J. Simpsonmurder trial:

•7 PCR loci in O. J. Simpson’s blood, found at the foyer ofNicole Brown’s apartment and leading out of the crimescene to Bundy Drive

•7 PCR loci in Nicole Brown’s blood, found in a pair ofsocks in Simpson’s bedroom

•2 PCR loci in Ronald Goldman’s blood, found on a pair ofbloody gloves at Simpson’s home