1. DNA Technology and Genomics

2. DNA Technology Methods for studying and manipulating genetic material
Cloning
Genetically Modified
Gene Therapy
DNA Profiling
Genomics

3. Recombinant DNA
Combining nucleotide sequences from 2 sources to form a single DNA
Bacteria often used
Plasmid

5. Creating Recombinant DNA
Restriction Enzymes
Restirction site
Sticky Ends
Ligase 5

6. Cloning the Gene Bacteria containing the gene are cloned
Typically, antibiotic resistance genes are also inserted
Bacteria are grown on antibiotic medium
All bacteria without the resistance (and target) gene die
Only those with gene reproduce
Stored in genomic library
Plasmids used to “store” genetic information

7. Uses Gene Cloning Genetic Engineering
Produces multiple copies of gene-carrying DNA
Genetic Engineering
Direct manipulation of genes

8. cDNA Express Eukaryotic genes in Prokaryotic cells mRNA
What about introns?
Prokaryotes don’t have machinery to splice
mRNA
Already spliced
Work from mRNA to create cDNA

9. and addition of reverse transcriptase; synthesis of DNA strand
Fig. 12-4
Cell nucleus
Exon
Intron
Exon
Intron
Exon
DNA of
eukaryotic
gene 1
Transcription
RNA
transcript 2
RNA splicing
mRNA 3
Isolation of mRNA
and addition of reverse
transcriptase; synthesis
of DNA strand
Test tube
Reverse transcriptase
cDNA strand
being synthesized 4
Breakdown of RNA 5
Synthesis of second
DNA strand
cDNA of gene
(no introns)

10. Table 12.6

11. Uses
Diagnosis and treatment of disease
Vaccines
Therapeutic hormones

12. Probes Genomic Library Nucleic Acid Probe How to find the right gene?
Locate specific gene or nucleotide sequence

13. Probes
Synthesize short sequence of singe stranded DNA of complimentary sequence
Label radioactive tag
Mix with genomic library
Bacteria with gene of interest will glow

14. Radioactive DNA probe Mix with single- stranded DNA from
Fig. 12-5
Radioactive
DNA probe
Mix with single-
stranded DNA from
genomic library
Single-stranded
DNA
Base pairing
indicates the
gene of interest

15. Genetically Modified
Organisms who have acquired genes by artificial means
If from another species= transgenic organism
Agrobacterium tumefaciens
Plant cell
DNA containing
gene for desired trait 1 Ti
plasmid
Recombinant
Ti plasmid 2 3
Introduction
into plant
cells
Regeneration
of plant
Insertion of gene
into plasmid
DNA carrying new gene
Plant with new trait
Restriction site

16. Gene Therapy
Altering an afflicted persons genes for therapeutic purposes
Treatment of genetically based disorders
Technology still in infancy

17. Cloned gene (normal allele) Insert normal gene into virus
Fig
Cloned gene
(normal allele) 1
Insert normal gene
into virus
Viral nucleic acid
Retrovirus 2
Infect bone marrow
cell with virus 3
Viral DNA inserts
into chromosome
Bone marrow
cell from patient
Bone
marrow 4
Inject cells
into patient

18. DNA Profiling Forensics DNA Profiling Scientific analysis of evidence
Analysis of DNA fragments
Determine if they originate form a particular individual
PCR
Gel Electrophoresis

19. PCR 3 step cycle Heat separates strands
Cooling allows primers to form H bonds with end of target sequence
DNA Polymerase adds nucleotides
Creates double stranded DNA
Repeat

20. PCR Significant breakthrough in genetics
Original polymerase was from E. coli
Heating traditionally denatured polymerase
New polymerase added after each heating cycle
Giant pain in the butt

21. PCR Discovery of T. aquaticus Could now be done at higher temps
Lives in hotsprings
Not denatured under high temps
Could now be done at higher temps
Better success rates

22. PCR Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules Cycle 3
Genomic
DNA 3 5 3 5 3 5 5 1
Heat to
separate
DNA strands 2
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequences 3
DNA
polymerase adds
nucleotides
to the 3 end
of each primer 3 5 5 3
Target
sequence 5 5 3 5 3 5 3
Primer
New DNA

23. Gel Electrophoresis
Use of gel to separate DNA strands by size (molecular weight) or charge
DNA must first be “digested”
Strands must be cut into different sizes
Use Restriction Enzymes
Cut DNA in specific places
Looks for specific nucletoide sequences

24. Gel Electrophoresis – + Mixture of DNA molecules of different sizes
Power
source
Gel
Mixture of DNA
molecules of
different sizes
Longer
molecules
Shorter
Completed gel
Figure 12.10

25. Gel Electrophoresis STR Create a genetic profile of individuals
Short sequences of DNA repeated many times in a row
STR analysis compared lengths of STR sequences at specific sites on the genome
Create a genetic profile of individuals
STR’s differ among individuals

27. Gel Electrophoresis RFLP Restriction fragment length polymorphisms
Difference between two samples of homologous DNA arising from differing locations of restriction sites

28. Crime scene
Suspect w x y z
Cut
DNA from chromosomes C G A T

29. After digestion by restriction enzymes the fragments are run through a gel– +
Longer
fragments
Shorter x w y z 1 2
Figure 12.11B

30. DNA profiling Crime scene Suspect 1 Suspect 2 DNA isolated
DNA of selected
markers amplified
Amplified DNA
compared 3

31. Used in forensic investigations
STR site 1
STR site 2
Crime scene DNA
Number of short tandem
repeats match
Number of short tandem
repeats do not match
Suspect’s DNA

32. Genomics
Studying the entire genome and their interactions

33. Human Genome Project
identify all the approximately 20,000-25,000 genes in human DNA,
determine the sequences of the 3 billion chemical base pairs that make up human DNA,
store this information in databases,
improve tools for data analysis,
transfer related technologies to the private sector, and
address the ethical, legal, and social issues (ELSI) that may arise from the project.

34. Exons (regions of genes coding for protein
Fig
Exons (regions of genes coding for protein
or giving rise to rRNA or tRNA) (1.5%)
Repetitive
DNA that
includes
transposable
elements
and related
sequences
(44%)
Introns and
regulatory
sequences
(24%)
Unique
noncoding
DNA (15%)
Repetitive
DNA
unrelated to
transposable
elements
(15%)

35. HGP 3.2 billion nucleotide pairs RNA splicing Non-coding
~21000 genes
How are we so complex?
RNA splicing
Non-coding
Repetitive DNA
Telomeres
Transposable elements

36. Shotgun Method Chop up genome Clone fragments Sequence
Squeeze it through a small, pressurized syringe
Clone fragments
Insert them into a vector
Sequence
Search for overlapping segments
Reassemble the overlaps

37. Chromosome
Chop up with
restriction enzyme
DNA fragments
Sequence
fragments
Align
fragments
Reassemble
full sequence

38. Recombo DNA
PCR
PCR and Gel

39. Videos
Ch Overview