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Students
- Welcome back!
- Get test folders from table
- Grades due tomorrow at lunch
- Missing items
- Unit 5 test
- Lab notebooks
- Today – Review Mid-term – Curve set later today
- Avg: 27 (out of 36) 75%
- Range: 15-35
- Still working on grades
- Syllabus coming on Monday….diverge from Mercer’s group
- Phones in bins…off or muted…please and thank you
AthElites 2015 National Champions!!
Students
- Get 2 handouts
- Syllabus
- Biotech pre-test
End times
4th 12:45
7th 2:40
8th 3:10
- Get test folders & lab notebooks
- 2nd quarter grades posted
Semester
avg
1st
2nd
4th
7th
8th
79.9
81.9
85.3
85.1
81.6
- New seats today…say good bye & then hello
- Needed – volunteers for tomorrow night’s Open House
- After moving, phones in bin…muted or off…please & thank you
Chapter 20 Essential Question
LO 3.5 The student can justify the claim that humans can manipulate heritable
information by identifying at least two commonly used technologies.
Figure 20.2 Overview of gene cloning
Bacterium
1 Gene inserted
Cell containing gene
of interest
into plasmid
Bacterial
chromosome
Gene of
interest
Plasmid
Recombinant
DNA (plasmid)
2 Plasmid put into
DNA of
chromosome
bacterial cell
Recombinate
bacterium
3 Host cell grown in culture,
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein expressed
by gene of interest
Protein harvested
4 Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth
hormone treats
stunted growth
Chapter 20: DNA Technology and Genomics
1. How is a gene cut out of a chromosome?
- Restriction enzymes
Restriction site
- Recognize a palindrome sequence
GAATTC
- Originally found in bacteria
DNA 5
CTTAAG
3
- Overhangs are “sticky ends” &
enzyme cuts
will bind to any complementary 1 Restriction
the sugar-phosphate
backbones at each arrow
sequence
- DNA ligase makes a recombinant
G
G
DNA molecule
3
5
Sticky end
2 DNA fragment from
another source is added.
Base pairing of sticky
ends produces various
combinations.
G AATTC
C TTAA G
G
G
Fragment from different
DNA molecule cut by the
same restriction enzyme
G AATTC
CTTAA G
One possible combination
3 DNA ligase
seals the strands.
Recombinant DNA molecule
Students
- Open House volunteers
- 6–8
- 20’ increments
- Books for a library in Ghana…in lieu of cans
- New or gently used
- Pre-school – 6th grade
- Atkins Key Club
- Phones in bin…off or muted…please & thank you
Mr. Bennett,
I know it has been a while, but this is Hope Kelly and I took your AP
biology class my junior year of high school at the Career Center. I
currently am a sophomore at NC State and have decided to major in
human biology with a plan to apply to a graduate program for physical
therapy upon graduation. I have been meaning to reach out to you for a
while to tell you how appreciative I am to have taken your course during
high school. I can not express to you how prepared I have been for my
biology classes here at State due to your AP Biology course and I just
wanted to let you know what a great teacher and mentor you were to
me! I am currently taking a genetics course this semester and I still
remember concepts that you taught in your class that continue to help
me in my studies. I remember your class being one of the hardest of
my high school career, but I now appreciate the vigor and passion you
incorporate into your course material and teaching practices. I hope
that all is well and that you are having a great start to your semester!
Chapter 20: DNA Technology and Genomics
1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?
1 Isolate plasmid DNA and human DNA.
Bacterial cell
lacZ gene (lactose breakdown)
Human cell
Restriction
site
ampR gene
(ampicillin
resistance)
2 Cut both DNA samples with the same restriction
enzyme, one that makes a single cut within the
lacZ gene and many cuts within the human DNA.
3 Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
Fragments
Recombinant DNA plasmids
Chapter 20: DNA Technology and Genomics
1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?
1 Isolate plasmid DNA and human DNA.
Bacterial cell
lacZ gene (lactose breakdown)
Human cell
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid
Sticky
ends
2 Cut both DNA samples with the same restriction
enzyme, one that makes a single cut within the
lacZ gene and many cuts within the human DNA.
3 Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
4 Introduce the DNA into bacterial cells that have a
mutation in their own lacZ gene.
Gene of
interest
Human DNA
Fragments
Recombinant DNA plasmids
Recombinant
bacteria
Chapter 20: DNA Technology and Genomics
1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?
1 Isolate plasmid DNA and human DNA.
Bacterial cell
lacZ gene (lactose breakdown)
Human cell
Restriction
site
ampR gene
(ampicillin
resistance)
Bacterial
plasmid
Sticky
ends
2 Cut both DNA samples with the same restriction
enzyme, one that makes a single cut within the
lacZ gene and many cuts within the human DNA.
3 Mix the DNAs; they join by base pairing.
The products are recombinant plasmids
and many nonrecombinant plasmids.
4 Introduce the DNA into bacterial cells that have a
mutation in their own lacZ gene.
5 Plate the bacteria on agar containing
ampicillin and X-gal. Incubate until
colonies grow.
Gene of
interest
Human DNA
Fragments
Recombinant DNA plasmids
Recombinant
bacteria
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying recombinant plasmid
with disrupted lacZ gene
Bacterial
clone
Chapter 20: DNA Technology and Genomics
1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?
3. How are genomes of interest kept in a research lab?
- Genomic libraries
- Collection of clones in either plasmids or phages
Foreign genome
cut up with
restriction
enzyme
or
Recombinant
plasmids
Bacterial
clones
(a) Plasmid library
Recombinant
phage DNA
Phage
clones
(b) Phage library
Chapter 20: DNA Technology and Genomics
1.
2.
3.
4.
How is a gene cut out of a chromosome?
How is recombinant DNA cloned?
How are genomes of interest kept in a research lab?
How can we find a “gene of interest” in a genomic library?
- Screen a genomic library using a radioactive probe
- Nucleic acid probe hybridization
Figure 20.5 Nucleic acid probe hybridization
Chapter 20: DNA Technology and Genomics
1.
2.
3.
4.
5.
How is a gene cut out of a chromosome?
How is recombinant DNA cloned?
How are genomes of interest kept in a research lab?
How can we find a “gene of interest” in a genomic library?
What is cDNA & how is it made?
- complementary DNA
- complementary to mRNA
- Only exons present
- Isolate mRNA
- Use reverse transcriptase to make cDNA
- cDNA libraries made from different tissues, stages of development,
in response drugs, etc
Students
- Thank you for those who came out last night.
-
Phone in bin…muted or off…please & thank you!
Chapter 20: DNA Technology and Genomics
1.
2.
3.
4.
5.
6.
How is a gene cut out of a chromosome?
How is recombinant DNA cloned?
How are genomes of interest kept in a research lab?
How can we find a “gene of interest” in a genomic library?
What is cDNA & how is it made?
What is PCR & how is it used?
- Polymerase chain reaction
- Used to amplify DNA
- Forensics
- Paternity testing
Figure 20.7 The polymerase chain reaction (PCR)
Making DNA
- Template
- Primers
- dNTPs
- DNA polymerase (Taq – heat resistant)
1. Denature DNA – 95°C
2. Annealing – 65°C
3. Extension – 72°C
Repeat this cycle 25 – 35 times
Each cycle doubles the DNA
Chapter 20: DNA Technology and Genomics
1.
2.
3.
4.
5.
6.
7.
How is a gene cut out of a chromosome?
How is recombinant DNA cloned?
How are genomes of interest kept in a research lab?
How can we find a “gene of interest” in a genomic library?
What is cDNA & how is it made?
What is PCR & how is it used?
What is gel electrophoresis?
- Method to separate DNA, RNA or protein based on size & charge
- Forest analogy….
Figure 20.8 Gel Electrophoresis
1.
2.
3.
4.
5.
DNA loaded into wells
Electrical current applied
(-) DNA moves toward (+)
Shorter molecules move faster
DNA is visualized
Chapter 20: DNA Technology and Genomics
1.
2.
3.
4.
5.
6.
7.
8.
How is a gene cut out of a chromosome?
How is recombinant DNA cloned?
How are genomes of interest kept in a research lab?
How can we find a “gene of interest” in a genomic library?
What is cDNA & how is it made?
What is PCR & how is it used?
What is gel electrophoresis?
What is RFLP analysis?
- Restriction Fragment Length Polymorphism
- Illustrates each person has a unique DNA fingerprint (1st forensic test)
- Combines restriction digestion & gel electrophoresis
Figure 20.9 Using restriction fragment analysis to distinguish
the normal and sickle-cell alleles of the -globin gene
Normal  -globin allele
201 bp
175 bp
DdeI
DdeI
DdeI Recognition Site
Large fragment
DdeI
DdeI
Sickle-cell mutant -globin allele
Large fragment
376 bp
Ddel
Ddel
Ddel
(a) DdeI restriction sites in normal and sickle-cell alleles of
-globin gene.
Normal Sickle-cell
allele
allele
Large
fragment
376 bp
201 bp
175 bp
(b) Electrophoresis of restriction fragments from
normal and sickle-cell alleles.
Chapter 20: DNA Technology and Genomics
DNA Forensics now
- Uses PCR to amplify DNA at 13 different loci
- Short Tandem Repeats (STRs) are amplified
- …CCCTCATTCATTCATTCATTCATCGC…
-
CODIS – Combined DNA Index Service – federal DNA database
Using 13 loci increases discrimination & reduces Random Match Probability
RMP = ~1 in 2.7 trillion for each of us
Chapter 20: DNA Technology and Genomics
1.
2.
3.
4.
5.
6.
7.
8.
9.
How is a gene cut out of a chromosome?
How is recombinant DNA cloned?
How are genomes of interest kept in a research lab?
How can we find a “gene of interest” in a genomic library?
What is cDNA & how is it made?
What is PCR & how is it used?
What is gel electrophoresis?
What is RFLP analysis?
What is Southern blot analysis?
- Combination of RFLP & nucleic acid probe hybridization
- Transfers DNA from gel to a solid substrate (nitrocellulose paper)
Figure 20.10 Southern blotting of DNA fragments
Students
- Get possible FRQs
- Techniques covered
- Restriction enzymes
- Making recombinant DNA
- Cloning & transformation
- Screening a library
- cDNA
- PCR
- RFLPs (early & current forensics)
- Southern blotting (northern & western)
-
Learning logs….11 questions….start NOW!!!!
-
Phones in bin….off or muted…please & thank you
Chapter 20: DNA Technology and Genomics
1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?
3. How are genomes of interest kept in a research lab?
4. How can we find a “gene of interest” in a genomic library?
5. What is cDNA & how is it made?
6. What is PCR & how is it used?
7. What is gel electrophoresis?
8. What is RFLP analysis?
9. What is Southern blot analysis?
10. What is a northern blot & a western blot
- northern – detects RNA with nucleic acid probe
- western – detects protein with an antibody
11. How is DNA sequenced?
- Dideoxy termination method
- 3’ –OH is missing; therefore, no extension & termination occurs
- Combines copying DNA, electrophoresis, & fluorescent labeling
Figure 20.12 Dideoxy chain-termination method for
sequencing DNA
(10
:
Reagents needed to make DNA
+ dideoxy nucleotides (10:1)
What ever color is detected is the
last nucleotide.
No extension off of dideoxy nucleotide
1)
Chapter 20: DNA Technology and Genomics
1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?
3. How are genomes of interest kept in a research lab?
4. How can we find a “gene of interest” in a genomic library?
5. What is cDNA & how is it made?
6. What is PCR & how is it used?
7. What is gel electrophoresis?
8. What is RFLP analysis?
9. What is Southern blot analysis?
10. What is a northern blot & a western blot
11. How is DNA sequenced?
12. What are genomics?
- The study of whole sets of genes and their interactions
- Human Genome Project has provided sequence – now we must
determine how genes interact
- Proteomics – study of protein interactions
13. How can gene function be determined?
- in vitro mutagenesis – disable gene & observe consequences
- RNA interference (RNAi) – silencing of gene expression by making DSRNA with matching sequence which triggers breakdown of mRNA.
Chapter 20: DNA Technology and Genomics
1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?
3. How are genomes of interest kept in a research lab?
4. How can we find a “gene of interest” in a genomic library?
5. What is cDNA & how is it made?
6. What is PCR & how is it used?
7. What is gel electrophoresis?
8. What is RFLP analysis?
9. What is Southern blot analysis?
10. What is a northern blot & a western blot
11. How is DNA sequenced?
12. What are genomics?
13. How can gene function be determined?
- in vitro mutagenesis – disable gene & observe consequences
- RNA interference (RNAi) – silencing of gene expression by using DSRNA with matching sequence which triggers breakdown of mRNA.
14.What is a DNA microarray?
- Method used to measure expression of thousands of genes at once
- Uses cDNA to bind to gene segments on a glass slide
Figure 20.14 Research Method DNA microarray assay of
gene expression levels
Chapter 20: DNA Technology and Genomics
1. How is a gene cut out of a chromosome?
2. How is recombinant DNA cloned?
3. How are genomes of interest kept in a research lab?
4. How can we find a “gene of interest” in a genomic library?
5. What is cDNA & how is it made?
6. What is PCR & how is it used?
7. What is gel electrophoresis?
8. What is RFLP analysis?
9. What is Southern blot analysis?
10. What is a northern blot & a western blot
11. How is DNA sequenced?
12. What are genomics?
13. How can gene function be determined?.
14. What is a DNA microarray?
15. What are some practical applications of DNA technology?
- Disease diagnosis
- HIV diagnosis using RT-PCR
- PCR of “questionable” alleles followed by DNA sequencing
- Viral gene therapy
- Uses a virus to deliver a normal allele to “replace” a mutant allele
- Crossing over will “swap out” the alleles
Figure 20.16 Gene therapy using a retroviral vector
1. Insert “good” RNA into retrovirus
2. Infect bone marrow cells, which
are stem cells
3. Viral DNA inserts into chromosome
4. Inject “fixed” cells into patient
Fixing stem cells SHOULD have
lasting effects since they
can replicate themselves &
differentiate/mature.
Chapter 20: DNA Technology and Genomics
15. What are some practical applications of DNA technology?
- Disease diagnosis
- Viral gene therapy
- Production of pharmaceutical products
- Using expression vectors to make proteins
- Insulin, growth hormone, TPA
- Receptor mimic to block HIV infection
- Vaccine production
- Forensics
- Environmental Clean-up & Bioremediation
- Transgenic animals
- Transgenic plants
- Ti plasmid
Figure 20.19 Using Ti plasmid to produce transgenic plants