1. Chapter 12 DNA Technology and the Human Genome

2. Do Now: 10 minutes Study Technique
Skim chapter opening pg
1) Define bold words and look at pictures
2) Make a quick outline of main points with at least 3 spaces in between in one color
3) Use a different color to fill in under each section with details from the book
4) In a different color come up with two questions, one easy and one difficult

3. Should look like this….
Recombinant DNA technology: combining genes from different sources
DNA technology: methods for studying and manipulating DNA
Genomics: Study of genomes based on sequence
Bioinformatics: using math to analyze bio data
Proteomics: study of proteins
Bacteria studies
Joined by pilli (male) passing DNA to “female”
Respond to environment
Lederburg and Tatum: experiments that mixed genes in E. coli (Recombinant DNA technology
DNA technology
Introns discovered by this
Lead in to HGP
Human Genome project (more than just humans)
Mapping genomes lead to genomics, bioinformatics, and proteomics
Scientists engineer bacteria for practical purposes (make pesticides, cancer drugs, AIDS vaccine)
Ethical issues on health

4. What might Ms. Bell Ask… Easy: How do bacteria have sex?
Hard: Why is this advantageous for bacteria?
Easy: Describe the difference between genomics, proteomics, and bioinformatics
Hard: Why are people concerned with recombinant DNA technology? Explain a hypothetical situation.

5. 12.1 In nature, bacteria can transfer DNA in three ways
Gene transfer
DNA from one to another bacteria
Non-reproductive – new combination
DNA= closed loop w/protein
Three mechanisms
1) Transformation: uptake from surrounding
Griffith experiment: harmless bacteria take up piece from deadly and became deadly….
2) Transduction: phage
3) Conjugation: Male donor, pili
Female recipient
Cytoplasmic bridge
New DNA may integrate into recipient

6. Question… Which type of gene transfer allows one copy of DNA to peel off and transfer to recipient?

7. 12.2 Bacteria plasmids can serve as carriers for gene transfer
F factor
Genes for conjugation
Also contains origin of replication
Acts as the leading end of transferred chromosome
Plasmid
Small circular DNA that contains origin of replication
Some contain F factor
Vector s: Can carry genes other than F factor
R plasmid
Contain enzymes that destroy antibiotics like penicillin or tetracycline
Act as useful vectors in genetic engineering

8. How restriction enzymes work…
LE 12-02
Restriction enzyme
recognition sequence G A A T T C
How restriction enzymes work…
DNA C T T A A G
Restriction enzyme
cuts the DNA into
fragments G A A T T C C T T A A G
Sticky end A A T
Addition of a DNA
fragment from
another source T C G G C T T A A
Two (or more)
fragments stick
together by
base-pairing G A A T T C G A A T T C C T T A A G C T T A A G
DNA ligase
pastes the strand
Recombinant DNA molecule

9. LE 12-01-3 Bacterium Plasmid isolated Cell containing gene of interest
DNA
isolated
Gene inserted
into plasmid
Bacterial
chromosome
Plasmid
Recombinant DNA
(plasmid)
Gene of
interest
DNA
Plasmid put into
bacterial cell
Recombinant
bacterium
Cell multiplies with
gene of interest
Copies of gene
Copies of protein
Clone of cells
Gene for pest
resistance
inserted into
plants
Protein used to
make snow
form at higher
temperature
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to dissolve blood
clots in heart attack therapy

10. 12.3 Plasmids are used to customize bacteria: An overview
1) plasmid isolated
2) DNA of interest is obtained
3) DNA inserted
4) Bacteria uptakes plasmid
5) genetically engineered, recombinant bacteria cloned
Challenge: How can R plasmids be helpful in terms of recombinant DNA technology?

11. LE 12-03 E. coli Human cell Isolate DNA from two sources Cut both DNAs
with the same
restriction enzyme
Plasmid
DNA
Gene V
Sticky ends
Mix the DNAs;
they join by
base-pairing
Add DNA ligase
to bond the DNA covalently
Recombinant DNA
plasmid
Gene V
Put plasmid into bacterium
by transformation
Recombinant
bacterium
Clone the bacterium
Bacterial clone carrying many
copies of the human gene

12. Genome cut up with restriction enzyme Recombinant plasmid Recombinant
LE 12-04
Genome cut up with
restriction enzyme
Recombinant
plasmid
Recombinant
phage DNA or
Bacterial
clone
Phage
clone
Plasmid library
Phage library

13. 12.7 Reverse Transcriptase helps make genes for cloning
Do Now: Turn to page 236 section 12.7
1) Read the section
2) Redraw the picture
3) Describe cDNA and the process of making a cDNA
4) Why might cDNA of insulin might work better in E.coli than trying to use a normal gene?

15. LE 12-05 Cell nucleus Exon Intron Exon Intron Exon DNA of eukaryotic
gene
Transcription
RNA
transcript
RNA splicing
(removes introns)
mRNA
Isolation of mRNA
from cell and addition
of reverse transcriptase;
synthesis of DNA strand
Test tube
Reverse transcriptase
cDNA strand
Breakdown of RNA
Synthesis of second
DNA strand
cDNA of gene
(no introns)

16. 12.8 Nucleic acid probes identify clones carrying specific genes
It is difficult to tell if clone contains ________________________
Methods depends on base pairing
Part of the DNA is known
Gene _________
Probe (radioactive) _________
Steps:
1) _________________________________
2) Treat (heat or alkali) to ___________________
3) Add radioactive probe (reality ___________________)
4)Autoradiography (develop film to determine radioactivity)
5) Compare to master plate (____________________)

17. Radioactive probe (DNA) A A T C C G Mix with single- stranded DNA from
various bacterial
(or phage) clones
Single-stranded
DNA G C T A A G T T C T A C C C A G T A G C T A G T C T A G A T G A T T C G
Base pairing
indicates the
gene of interest C G G A A

18. 12.9 DNA microarrays test for expression of many genes
DNA microarrays (chips)
Used for______________________________
Measures gene expression at _______________
Steps
______________________
mRNA used to make radioactive ________
Exposed to a chip
Benefits
Thousands of genes tested at once
Each radioactive spot represents gene expression

19. LE 12-09 DNA microarray Each well contains DNA from a particular gene
Actual size
(6,400 genes)
mRNA
isolated
Unbound
cDNA rinsed
away
Reverse transcriptase
and fluorescent DNA
nucelotides
Fluorescent
spot
Nonfluorescent
spot
cDNA applied
to wells
cDNA made
from mRNA
cDNA
DNA of an
expressed gene
DNA of an
unexpressed gene

20. 12.10- 12.11 Gel electrophoresis:
Mixture of DNA
molecules of
different sizes
Longer
molecules
Power
source
Gel
Shorter
molecules
Completed gel

21. 12.12 The PCR method is used to amplify DNA sequences
Polymerase chain reaction
Steps:
_____________________
DNA is incubated with____________________________________________
Benefits:
Each cycle doubles DNA amount
Takes hours
Can copy specific segment
Starting material does not need to be purified

22. PCR: The good and the bad
Even though it is faster, it cannot replace gene cloning in cells due to occasional errors that impose limits
Uses:
________________________

23. 12.13 Most of the human genome does not consist of genes
Most of our DNA does not code for protein
35,000 genes that code for protein, tRNA and rRNA
97% is non-coding

24. Types of junk __________and DNA between genes Repetitive DNA
Short repeats
Associated with ________________and ends of chromosomes (chromosome structure)
Telomeres: at the end of chromosomes and related to cell death and cancer
Cells that can regenerate telomeres can “_______________”
Might deal with gene ___________________
Some nervous diseases caused by abnormal stretches of triplet
Huntington's: _____ repeat in coding region
Long Repeats: function unknown
Example: Transposons: genes that can jump from one location to the next (researcher Barbara McClintock)
Cut and paste or ______, cut and paste (leaves sequence behind)
Natural mutagen to generate ________________
Implicated in some cancer

26. LE 12-11b 1 2
Longer
fragments z x w
Shorter
fragments y y

27. Blood from defendant’s clothes Defendant’s blood Victim’s blood
LE 12-12a
Blood from
defendant’s clothes
Defendant’s
blood
Victim’s
blood

28. LE 12-13 Cloned gene (normal allele) Insert normal gene into virus
Viral nucleic
acid
Retrovirus
Infect bone marrow
cell with virus
Viral DNA inserts
into chromosome
Bone marrow
cell from patient
Bone
marrow
Inject cells
into patient

29. LE 12-14
Initial
DNA
segment 1 2 4 8
Number of DNA molecules

30. Recombinant Lab
Add picture from each….