Transcript Ch_20

Chapter 20: DNA Technology and Genomics
- Lots of different techniques
- Many used in combination with each other
- Uses information from every chapter so far
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
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
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
- To aid in DNA sequencing
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 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
- Combines restriction digest & 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
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
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
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 PCR, electrophoresis, & fluorescent labeling
Figure 20.12 Dideoxy chain-termination method for
sequencing DNA
Reagents needed to make DNA
+ dideoxy nucleotides
What ever color is detected is the
last nucleotide.
No extension off of dideoxy nucleotide
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
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.
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