Chapter 20: DNA Technology and Genomics

- Lots of different techniques- Many used in combination with each other- Uses information from every chapter so far

Bacterium

Bacterialchromosome

Plasmid

Cell containing geneof interestGene inserted

into plasmid

RecombinantDNA (plasmid) Plasmid put into

bacterial cell

Gene of interest

DNA ofchromosome

Recombinatebacterium

Host cell grown in culture,to form a clone of cellscontaining the “cloned”gene of interest

Protein harvested

Basic research on protein

Basic research and various applications

Gene of interest

Copies of gene

Basic research on gene

Gene for pestresistance inserted into plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hormone treatsstunted growth

Protein expressedby gene of interest

1

2

3

4

Figure 20.2 Overview of gene cloning

Chapter 20: DNA Technology and Genomics

1. How is a gene cut out of a chromosome? - Restriction enzymes- Recognize a palindrome sequence- Originally found in bacteria- Overhangs are “sticky ends” &

will bind to any complementary sequence

- DNA ligase makes a recombinantDNA molecule

Restriction site

DNA 53 5

3G A A T T CC T T A A G

Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow

DNA fragment from another source is added. Base pairing of sticky ends produces various combinations.

DNA ligaseseals the strands.

Sticky end

Fragment from differentDNA molecule cut by thesame restriction enzyme

One possible combination

Recombinant DNA molecule

G

C T T A AA A T T C

G

A A T T C

C T T A AG

G

G GA A T T C A A T T C

C T T A A G C T T A A G

1

2

3

Chapter 20: DNA Technology and Genomics

1. How is a gene cut out of a chromosome?2. How is recombinant DNA cloned?

Recombinant DNA plasmids

Stickyends

Human DNA

Fragments

Human cell

Gene of interest

Bacterial cell

ampR gene(ampicillinresistance)

Bacterial plasmid

Restriction site

lacZ gene (lactose breakdown)1 Isolate plasmid DNA and human DNA.

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 plasmidsand many nonrecombinant plasmids.

Chapter 20: DNA Technology and Genomics

1. How is a gene cut out of a chromosome?2. How is recombinant DNA cloned?

Recombinantbacteria

Recombinant DNA plasmids

Stickyends

Human DNA

Fragments

Human cell

Gene of interest

Bacterial cell

ampR gene(ampicillinresistance)

Bacterial plasmid

Restriction site

lacZ gene (lactose breakdown)1 Isolate plasmid DNA and human DNA.

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 plasmidsand many nonrecombinant plasmids.

4 Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene.

Chapter 20: DNA Technology and Genomics1. How is a gene cut out of a chromosome?2. How is recombinant DNA cloned?

Colony carrying non-recombinant plasmid with intact lacZ gene

Bacterialclone

Colony carrying re-combinant plasmidwith disrupted lacZ gene

Recombinantbacteria

Recombinant DNA plasmids

Stickyends

Human DNA

Fragments

Human cell

Gene of interest

Bacterial cell

ampR gene(ampicillinresistance)

Bacterial plasmid

Restriction site

lacZ gene (lactose breakdown)1 Isolate plasmid DNA and human DNA.

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 plasmidsand 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 containingampicillin and X-gal. Incubate untilcolonies grow.

Chapter 20: DNA Technology and Genomics1. 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 genomecut up withrestrictionenzyme

Recombinantplasmids Recombinant

phage DNA Phageclones

(b) Phage library(a) Plasmid library

or

Bacterialclones

Chapter 20: DNA Technology and Genomics1. 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?

- Screen a genomic library using a radioactive probe- Nucleic acid probe hybridization

Figure 20.5 Nucleic acid probe hybridization

Chapter 20: DNA Technology and Genomics1. 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?

- 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 Genomics1. 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?

- Polymerase chain reaction- Used to amplify DNA- Forensics- Paternity testing- To aid in DNA sequencing

Figure 20.7 The polymerase chain reaction (PCR)

1. Denature DNA – 95°C2. Annealing – 65°C3. Extension – 72°C

Repeat this cycle 25 – 35 timesEach cycle doubles the DNA

Making DNA- Template- Primers- dNTPs- DNA polymerase (Taq – heat resistant)

Chapter 20: DNA Technology and Genomics1. 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?

- Method to separate DNA or protein based on size & charge- Forest analogy….

Figure 20.8 Gel Electrophoresis

1. DNA loaded into wells2. Electrical current applied3. (-) DNA moves toward (+)4. Shorter molecules move faster5. DNA is visualized

Chapter 20: DNA Technology and Genomics1. 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?

- Restriction Fragment Length Polymorphism- Combines restriction digest & gel electrophoresis

Normal -globin allele

Sickle-cell mutant -globin allele

175 bp 201 bp Large fragment

DdeI DdeI DdeI DdeI

Ddel Ddel Ddel

376 bp Large fragment

DdeI restriction sites in normal and sickle-cell alleles of -globin gene.

Electrophoresis of restriction fragments from normal and sickle-cell alleles.

Normalallele

Sickle-cellallele

Largefragment

201 bp175 bp

376 bp

(a)

(b)

Figure 20.9 Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the -globin gene

Recognition Site                  

Chapter 20: DNA Technology and Genomics1. 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?

- 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 Genomics1. 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

What ever color is detected is thelast nucleotide.No extension off of dideoxy nucleotide

Reagents needed to make DNA+ dideoxy nucleotides

Chapter 20: DNA Technology and Genomics1. 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 blot11. 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 interact13. How can gene function be determined?

- in vitro mutagenesis – disable gene & observe consequences- RNA interference (RNAi) – silencing of gene expression by using DS-

RNA with matching sequence which triggers breakdown of mRNA.

Chapter 20: DNA Technology and Genomics1. 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 blot11. 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 DS-

RNA 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