1. DNA Technology and Genomics
Chapter 14
DNA Technology and Genomics

2. Overview: Understanding and Manipulating Genomes
Sequencing of the human genome was largely completed by 2003
DNA sequencing has depended on advances in technology, starting with making recombinant DNA. Many are disappointed cures have not been found.
What organisms have been sequenced? Look here:

3. LE 20-2 Bacterium Cell containing gene of interest Gene inserted into
plasmid
Bacterial
chromosome
Plasmid
Gene of
interest
Recombinant
DNA (plasmid)
DNA of
chromosome
Plasmid put into
bacterial cell
Recombinant
bacterium
Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest
Protein expressed
by gene of interest
Copies of gene
Protein harvested
Basic research and
various applications
Basic
research
on gene
Basic
research
on protein
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Human growth hor-
mone treats stunted
growth

4. One possible combination Recombinant DNA molecule
Restriction site
DNA 5¢ 3¢ 3¢ 5¢
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
Sticky end
DNA fragment from another
source is added. Base pairing
of sticky ends produces
various combinations.
Fragment from different
DNA molecule cut by the
same restriction enzyme
One possible combination
DNA ligase
seals the strands.
Recombinant DNA molecule

5. LE 20-7
PCR – polymerase chain reaction. Making millions of copies of a portion of DNA to test. Thanks to bacteria from Yellowstone(with a heat stable DNA poly) – the reason forensics has evolved, sequencing the mammoth, Neanderthal etc.. We will do this on you!! 3 parts – heat DNA slightly to separate, cool so primers can bond, DNA poly adds nucleotides to 3” ends.
Primers
New
nucleo-
tides

6. LE 20-8 Mixture of DNA Longer molecules molecules of differ- Cathode
ent sizes
Longer
molecules
Cathode
Shorter
molecules
Power
source
Gel
Glass
plates
Anode

7. LE 20-9 Normal b-globin allele 175 bp 201 bp Large fragment Ddel Ddel
Sickle-cell mutant b-globin allele
376 bp
Large fragment
Ddel
Ddel
Ddel
Ddel restriction sites in normal and sickle-cell alleles of
-globin gene
Normal
allele
Sickle-cell
allele
Large
fragment
376 bp
201 bp
175 bp
Electrophoresis of restriction fragments from normal
and sickle-cell alleles RFLP – restriction length polymorphisms. Mutations that can be detected to see if you
have disease causing alleles.

9. Future Directions in Genomics
Genomics is the study of entire genomes. Same gene can make different proteins by splicing introns at different places!!
How do we know what a gene does? Knock it out and see what the consequences are.
Gene targeting is often used to inactivate single genes. Such gene 'knockout' experiments have elucidated the roles of numerous genes in embryonic development, adult physiology, aging and disease. To date, more than ten thousand mouse genes (approximately half of the genes in the mammalian genome) have been knocked out. Ongoing international efforts will make 'knockout mice' for all genes available within the near future.

10. Human Gene Therapy
Gene therapy is the alteration of an afflicted individual’s genes – like sickle cell.
Gene therapy holds great potential for treating disorders traceable to a single defective gene
Vectors are used for delivery of good genes into cells – like viruses which can easily fit receptors. Retroviruses make a DNA copy with the gene
Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

11. LE 20-16 Cloned gene Insert RNA version of normal allele
into retrovirus.
Viral RNA
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Retrovirus
capsid
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
Bone
marrow
Inject engineered
cells into patient.

12. Animal Husbandry and “Pharm” Animals
Transgenic organisms are made by introducing genes from one species into the genome of another organism
Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles)

13. Several organisms have similar genes that can be transferred between them. Ex: homeotic genes

14. Genetic Engineering in Plants
Agricultural scientists have endowed a number of crop plants with genes for desirable traits
The Ti plasmid is the most commonly used vector for introducing new genes into plant cells. Plants can produce a toxin from a bacteria, so when a caterpillar eats the leaf – its stomach explodes. Plants can also be “Round-up” resistant, so the weeds die from it but not the plants. Many plants world wide are now GMO’s – golden rice (w/ vitamin A for blindness) strawberries, tomatoes, corn etc…

15. LE 20-19
Agrobacterium tumefaciens Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
Recombinant
Ti plasmid
Plant with
new trait

16. Cloning – entire organism. A way to bring back or prevent extinctions?
Take DNA from a host cell (skin, hair) suck out DNA from an egg and put new DNA in.
Implant in a surrogate mother (must be related enough to not reject embryo). Wait until birth.
Dolly and many mammals have been cloned. Problems with disease and aging due to methylation of older DNA?? Genes not turning on as they do in infancy. Conclusion – clone in mammals not identical, not same personality

17. SNP – single nucleotide polymorphism
Spot where 1 base pair differs in at least 1% of the population. It’s a stable mutation so it helps track ancestry.