1. Recombinant DNA Technology
Methods for Isolating, Amplifying and Studying Specific DNA Sequences

2. Cutting and Joining DNA Fragments
Restriction Enzyme: cleaves DNA at specific sequences
DNA Ligase: joins DNA fragments by sealing nicks in backbone

3. Use of the Restriction Enzyme Bam H1
5’— G G A T C C — 3’
3’— C C T A G G — 5’
Results in
sticky end
5’— G G A T C C — 3’
3’— C C T A G G — 5’
DNA fragments with complementary sticky ends
can associate to form new DNA combinations.

4. Viewing DNA Fragments: Agarose Gel Electrophoresis

5. Locating DNA Fragments by Southern Blotting
Probe is complementary to DNA of interest
DNA Fragments are transferred to membrane
A subset of fragments binds to the probe

6. Cloning Genes
Gene cloning = isolation of a specific segment of DNA in a form that can be duplicated in another cell
Requires a
Vector = carrier of DNA segment
Host = cell in which foreign DNA can be duplicated and its gene product may be synthesized
Recombinant DNA = DNA produced by joining fragments from two or more different sources

7. Cloning Vectors For Bacterial Hosts Plasmid ampR Bam H1 tetR ampR
tetR gene disrupted, no longer functional

8. Cloning Vectors For Bacterial Hosts Bacteriophage Cosmid
Expression Vector

9. Cloning Vectors For Eukaryotic Hosts Shuttle Vector
YAC = yeast artificial chromosome
Ti plasmid

10. Tools for Finding Genes
DNA library = collection of clones from one DNA donor
Categorized by: vector, source of DNA
Genomic library = all of the sequences from the genome of a single organism
cDNA library= complementary DNA, made using mRNA as a template

11. Producing a Genomic Library
Source of DNA for Library
Many recombinant molecules are formed, each with a different DNA insert

12. Producing cDNA Primer binds to poly-A tail
DNA produced from RNA template
Partial digestion of RNA template
Second DNA strand produced using remaining RNA segments as primers
Gaps in backbone sealed

13. Tools for Finding Genes
Probe: sequence of DNA or RNA that is complementary to the gene of interest; Used to locate a copy of the gene in a library by hybridization
AATGCCAT
TTACGGTA
Denature DNA by heating
AATGCCAT
TTACGGTA
TTACGGTA
Add Probe
Probe Binds to gene

14. Screening a Library with a Probe

15. Amplifying DNA with the Polymerase Chain Reaction (PCR)
Copies a specific region in the DNA
Can be used with small amounts of sample
Uses repeated cycles of heating to denature DNA and cooling to synthesize new DNA
Requires
primers to begin synthesis
heat-stable polymerase (Taq polymerase)

16. Polymerase Chain Reaction

17. DNA Sequencing by the Sanger Method
Relies on Chain Termination during replication due to incorporation of Dideoxynucleotides OH H
deoxyribose H
dideoxyribose

18. Replicate unknown DNA using dideoxynucleotides to terminate growing chains at various positions
Unknown Sequence
3’-A-G-C-C-C-A-G-A-T-T-5’
Complementary Products
Black= dideoxynucleotide
5’-T-C-G-3’
5’-T-C-G-G-G-T-C-T-3’
5’-T-3’
5’-T-C-3’
5’-T-C-G-G-G-3’
5’-T-C-G-G-G-T-C-T-A-3’
5’-T-C-G-G-G-T-C-T-A-A-3’
5’-T-C-G-G-G-T-C-3’
5’-T-C-G-G-G-T-3’
5’-T-C-G-G-3’

19. Use gel electrophoresis to separate replicated fragments that differ by one base
A T C G
Dideoxynucleotide
Size of DNA on gel shows distance from start of replication
Labeling of dideoxynucleotide shows complementary sequence

20. Determine unknown sequence from complementary sequence with base-pairing rules
A T C G
Dideoxynucleotide
5’-T-C-G-G-G-T-C-T-A-A
5’-T-C-G-G-G-T-C-T-A
5’-T-C-G-G-G-T-C-T
5’-T-C-G-G-G-T-C
5’-T-C-G-G-G-T
5’-T-C-G-G-G
5’-T-C-G-G
5’-T-C-G
5’-T-C
5’-T
Complementary Sequence
Unknown Sequence

23. Applications of Recombinant DNA Technology
Genetic Testing and Gene Therapies
Products of Biotechnology

24. RFLP Analysis RFLP = Restriction Fragment Length Polymorphism
Identifies differences in the length of restriction fragments derived from similar DNA sequences
Analyzed by Southern Blotting
Used in gene mapping

25. RFLP Analysis in Paternity Testing

26. RFLP Analysis in Forensics1 2 3
C S R C I E
M N E E 4 5 6 7
Suspects
Suspects

27. DNA Fingerprinting
Detects patterns for short repeated sequences that are highly variable among humans
Described as
VNTR: Variable Number of Tandem Repeats
STR: Short Tandem Repeats
Microsatellites
Analysis using Microsatellite sequences

28. Genetic Testing

29. Gene Therapy
Andrew Gobea was born with SCID, an immune deficiency due to a defect in the ADA gene.
Four days after his birth, he received an injection of his own stem cells carrying functional ADA genes.
Andrew’s immune system began producing ADA, but the amount was not sufficient to maintain full health. He needs daily injections of ADA enzyme.

30. RNA Interference as a Possible Treatment for High Cholesterol
High levels of ApoB, a protein component of blood lipid carriers, are associated with high cholesterol levels
In monkeys, siRNA for ApoB has lowered the mRNA for ApoB in liver cells and also reduced serum cholesterol levels
siRNA for ApoB was delivered in lipid capsules

31. Recombinant DNA Products
Pharmaceutical
Used for
Factor VIII
Blood Clotting
Human Growth Hormone
Pituitary Dwarfism
Insulin
Diabetes
Interferon
Cancer
Tissue Plasminogen Activator
Heart Attack
Vaccine
Hepatitis B

32. Genetically Modified Organisms
Introducing or modifying specific genes to alter the phenotype of an organism
Transgenic: organism that contains a gene from another species in all of its cells
Transgenic Animals
Models of Human Disease
Produce Pharmaceuticals

33. Studying Gene Function with Knockout Mice
neo+ gene gives resistance to G418
tk+ gene gives sensitivity to gancyclovir
Insertion of neo+ into target gene yields neo+ and tk- cells, resistant to G418 and to gancyclovir

34. Transgenic Plants “Golden” rice with beta-carotene and extra iron
Round Up Ready Soybeans are resistant to herbicide
Bt Corn produces its own pesticide

35. What Are Some Concerns?