Genetic Engineering and

Recombinant DNA

Chapter 10

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Learning Objectives:• Define restriction enzymes and outline how they are used

to make recombinant DNA.

• List the properties of vectors. Outline the process of genetic cloning.

• List at least five applications of recombinant DNA technology

• Explain the purpose and identify the steps in polymerase chain reaction (PCR).

• Describe microarray technique and list its applications.

• List and describe at least five applications of microbial and human genomics

Applications of Genetic Engineering

• Artificial manipulation of genetic information

• Characterize organisms

• Indentify organisms

• Modify organisms

• More useful organisms

• Produce useful products

DNA strands can be cut across

• Enzymes that cut DNA

• Each recognizes a known sequence of 4 to 10 base pairs

• The sequences are usually palindromes (Madam, I’m Adam)

Some Common Restriction Enzymes

• BamHI - Bacillus amyloliquefaciens G|GATCCCCTAG|G

• EcoRI - Escherichia coli G|AATTCCTTAA|G

• EcoRV - Escherichia coli GAT|ATCCTA|TAG

DNA fragments can be joined together

• Restriction fragments.

• Ligase rejoins the ends of restriction fragments by forming sugar-phosphate bonds

The required gene is isolatedfrom a genetic donor.

Cloning vector (plasmid) iscommercially acquired.

Gene and plasmid are exposed to the same restriction endonuclease,producing complementaryends.

Complementary ends bind to form a single recombinant plasmid.

Genetic Cloning

Cloning Human Insulin Gene

1. Isolate human insulin gene

2. Splice into a cloning vector

3. Insert recombinant plasmid in bacterial cells

4. Screen for recombinant plasmids

5. Identify clones carrying human insulin gene

HindIIISphIPstISalIXbaIBamHISmaIKpnISacIEcoRI

MCS

pUC19(2,686bp)

Am

pR

ori

lacZ

Cloning Vectors

• Accept foreign DNA

• Able to “self-replicate”

• Need a promoter

• Selectable markers

• Examples:

• Plasmids

• Bacteriophage

• Phagemids

• Cosmids

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Bacteria withrecombinantplasmid

Ampicillin-resistance gene

Pure cultureof bacteriacontainingcloned gene

Selective medium

with ampicillin

Regular nonselectivemedium with twotypes of colonies

Bacteria carryingplasmid

Bacteria lackingplasmid

Culture of cloninghost after incubationwith recombinantplasmid

(2)(1)

Transformation: Selecting Clones

• Use selectable marker

• Selective media

• E.g. antibiotics

• Differential media

• E.g. X-gal

• Indicates inserts

Transformation. The recombinant plasmidis introduced into a cloning host cell.

The transformed cell is grown in culture to increase numbers. Every cell will contain a replicated plasmid containing the desired gene.

Cells transcribe and translate the foreign gene.

This yields a protein product that is recovered from the culture and purified.

Genetic Cloning

Human, other mammal,microorganism, or plant cell gene identified.

DNA of interest is isolated.

DNA of interest is insertedinto a cloning vector.

Cloning host receivesvector, becomes arecombinant

Cloning host provides abundant DNA for study.

Cloning host translates foreignDNA into protein.

Pharmaceutical proteins• insulin• human growth hormone

Vaccines• hepatitis B

Altered organisms with economically useful traits: transgenic plants• pest resistance• herbicide resistance• improved nutritional value

Recombinant DNAStrategies

• Obtain target gene

• Insert into vector

• Introduce into hosts

• Expression of desired gene product

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Gene Therapy

• Some diseases due to the lack of a protein

• Introduce good protein

• Phenotype corrected

1

2

3

7

6

5

4

Marrow cell7.Patient is observed for expression of normal gene.

6. Transfected cells are reinfusedinto patient.

5. Marrow cells are infected withretrovirus.

4. Bone marrow sample is taken from patient with genetic defect.

3. Gene is inserted into retrovirus vector.

2. Gene is cloned.

1. Normal gene is isolated fromhealthy subject.

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Genetic Probes

• Short single-stranded DNA sequences

• Identity specific target sequences

• Bound markers

• Dye

• Radioisotope

• Enzyme

Polymerase Chain Reaction

• Small amount of DNA or RNA limit some tests

• Polymerase chain reaction (PCR) rapidly increases the amount of DNA in a sample

• Can amplify a target DNA from a few copies to billions in a few hours

• Can detect a single cell

Three Basic Steps that Cycle• Denaturation

• Heat to 94°C to separate in to two strands

• Cool to between 50°C and 65°C

• Priming

• Primers added that are complementary to the target

• Extension

• 72°C

• DNA polymerase and nucleotides are added

• Polymerases extend the molecule

• Repeat about 25 to 30 times

Polymerase Chain Reaction

18

Microarrays

Microarray Applications

• Gene expression

• Organism detection

• Phylogenetic relationships

Microbial and Human Genomics

• Many microbial genomes have been sequenced

• Segments of the human genome have “microbial ancestors

• Safer food production

• Identification of uncultured microorganisms

• Microbial forensics

• New perspective to defining infectious diseases and studying evolution