1. Chapter 7 Recombinant DNA Technology and Genomics

2. Recombinant DNA DNA which has been recombined Gene cloning
multiple copies of a single gene

3. Gene Cloning Obtaining DNA Splicing genes into cloning vector
Putting recombinant DNA into host cell
Finding the right gene
Propagating host cell to produce clones of cells that carry the clone of gene

4. Gene Cloning
Figure 7.1

5. Gene cloning: Obtaining DNA
Precipitate DNA in alcohol
Wrap on glass rod or centrifuge
Dissolve in water or buffer
Figure 7.2

6. Cloning eukaryotic genes
Eukaryotic DNA has noncoding regions
introns
Obtain mRNA
introns are removed
Reverse transcribe
make DNA copy of the mRNA
Figure 7.3

7. Gene cloning: Cutting DNA
Restriction endonucleases (RE)
natural products of bacteria
protect from foreign viral DNA
cut DNA at specific sequences
select RE that cuts outside the gene of interest
also cut the vector (plasmid) with same RE 5’
G A A T T C 3’
C T T A A G 3’ 5’
EcoRI G
A A T T C 5’ 3’ 5’ 3’
C T T A A G 3’ 5’ 3’ 5’

8. Gene cloning: Gel electrophoresis
agarose gel
apply electrical current
DNA separates by size (length)
negative charge on DNA
moves toward positive electrode
small move faster than larger
isolate DNA containing gene
cut out of gel and remove from agarose

9. Gene cloning: Ligation
combine fragments of DNA
DNA containing gene
DNA containing vector (plasmid)
both cut with restriction endonuclease
add DNA ligase
binds DNA fragments together

10. Gene cloning: Transformation
Put recombinant DNA into host cell
recombinant DNA using plasmids
Artificial transformation
transform E. coli
treat with CaCl2 on ice
add recombinant DNA
heat shock
grow bacteria and screen for transformed cells

11. Gene cloning: Transduction
Recombined DNA in a phage vector
Use virus to inject recombined DNA into host bacteria
Grow up bacteria for cloned gene production
Figure 7.5

12. DNA libraries genomic library cDNA library chromosomal DNA
digest or shear
clone into vectors
screen for gene of interest or save for future use
cDNA library
isolate mRNA
make cDNA (complementary or copy DNA)

13. cDNA cloning mRNA isolated from cell mRNA
reverse transcriptase and dNTPs
cDNA
DNA polymerase and dNTPs
dsDNA
ligate into plasmid vector

14. Finding DNA using a Probe
Figure 7.7

15. Amplify Genes Importance Polymerase Chain Reaction (PCR)
amplify a specific section of DNA
primers specific for DNA section
allow DNA polymerase to make copies
result is billions of copies
Importance
Gene cloning: source of specific DNA
Forensic science: small amount of DNA
Medical Diagnosis: small quantities needed

16. Polymerase Chain Reaction
Necessary components
DNA to be used as template
Two primers: complementary regions that border the DNA to be amplified
Heat stable DNA polymerase
deoxynucleside triphosphates
specific buffer conditions
Cycle through heating and cooling
heat: denature two strands
cool: primers attach and polymerase copies template
repeat cycle--exponential increase in strands of DNA

18. Genomics Understanding organisms based on DNA sequence Sequencing
Use of dideoxynucleotides
Terminates DNA elongation
Electrophoresis of varying length fragments
Determines sequence
Automated
Each nucleotide labeled with different fluorescent marker
Laser causes fluorescence and detector determines nucleotide
Figure 7.10

19. Genomics Microarray technology
Compare expression of genes under certain conditions
Sporulating
Non-sporulating
Figure 7.11

20. Genomics Yellow Green Red Expression under both conditions
Nonsporulating expression
Red
Sporulating expression
Figure 7.12