Chapter 20

DNA Technology & Genomics

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Biotechnology Terms

Biotechnology Process of manipulating organisms or their components to

make useful products Genetic engineering + tissue/cell culturing technologies

Genetic Engineering Manipulation of individual genes or entire genomes Insulin (insulin E. coli bacteria OR yeast) & GMO

(Genetically Modified Organism)

Recombinant DNA Artificially created DNA Typically, DNA is integrated from another species

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Biotechnology Terms (Page 2)

Gene Cloning Laboratory production of multiple copies of DNA segment Therapeutic cloning – embryonic stem cells

Spinal cord injuries Reproductive (organismal) cloning – Dolly the sheep

Restriction Enzymes Enzymes that cut DNA at specific locations Usually, derived from bacteria Cut sites of DNA = restriction fragments Sticky ends – restriction fragments usually have one end

longer than the other

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Quick Assignment

Relate the 6 terms just discussed in a concept map.

Be prepared to defend your arrangement

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Cloning Process

5 steps (first 2)1. Identify & isolate the gene of interest

Involves finding a cloning vector – plasmid or organism used to carry the DNA sequence to be cloned

2. Cut gene of interest from original site & open up vector’s DNA using a ________ ________ This ensures matching sticky ends on gene of interest &

vector DNA

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Cloning Process (Page 2)

5 steps (3-4)3. Combine the 2 DNA pieces (into a recombinant plasmid?)

Recombinant plasmid – plasmid + DNA fragments Sealed together using DNA Ligase Remember: we used ________ ________ to cut gene of

interest from original site & cut vector’s DNA This ensures matching sticky ends on gene of interest &

vector DNA

4. Transfer the vector (recombinant plasmid) into a host cell Usually involves bacterial transformation

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Bacteria & Genetic Recombination

Conjugation Bacterial Sex Genetic material is exchanged

by direct contact

Transduction Phage transfer of DNA Involves a phage vector

Phage moves the DNA from bacterium to other bacterium

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Bacteria & Genetic Recombination

Transformation Uptake of exogenous

DNA Griffith’s experiment -

pathogenic DNA was transferred to benign bacteria

Most common method for genetic engineering

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Step 5

Select for transformed cells Link the gene of interest with

a reporter gene Such as pBLU or pGLO pBLU = Blue coloration pGLO = fluorescent green

under UV light

In Lab 6, we will insert the coloration gene and an ampicillin resistance gene to select for transformed cells

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At this point…

You know which cells have the gene of interest

You can identify the cells that have the gene of interest

Now what?

You need to extract the gene of interest

How would you do that?

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Nucleic Acid Hybridization

Detects the gene of interest

Uses a short, single stranded DNA or RNA called a nucleic acid probe

The nucleic acid probe is complementary to a known sequence in the gene of interest

Usually attach a radioactive isotope or fluorescent tag protein so that it is detectable

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Genomic Libraries

Nucleic Acid Hybridization repeated many times produces a genomic library Thousands of recombinant clones Each has a piece of the original genome being studied

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cDNA Library

cDNA = complementary DNA

mRNA is extracted from cells

Use what enzyme to make DNA from this mRNA?

Then make another strand of DNA using what enzyme?

cDNA library is only a portion of the genome Portion that codes for mRNA Exons? Introns? tRNA? rRNA?

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Microarray Assay

Genome-wide study of gene expression

Different genes are in each well

Identifies gene interactions + provides clues to gene functions

Take samples throughout development + assay to determine which genes are expressed and at what stages Detect patterns of expression throughout development Detect likely response to a pathogenic agent

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PCR Polymerase Chain Reaction Thermal cycling Amplification of DNA

3 Steps Denaturation (Heating)

Annealing (Cooling)Primer formation

Extension DNA polymerase adds nucleotides at 3’ end

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Gel Electrophoresis DNA is negatively charged so it moves AWAY from the (-) cathode toward the (+) anode

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Southern Blotting

Used to detect specific DNA sequences

Useful for comparing samples

Combines gel electrophoresis + nucleic acid hybridization

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DNA Technology affects us…

Disease Diagnosis PCR used to detect traces of viral DNA or RNA in sample RFLP (Restriction Fragment Length Polymorphisms)

Different alleles have different RFLPs

Gene Therapy – alter afflicted genes

Pharmaceutical Production – Insulin production

Forensic Application – DNA fingerprints

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Environmental cleanup Genetically engineered microbes Detoxification of specific wastes

Agricultural applications Insert pest-resistant or drought-resistant genes GMO (Genetically Modified Organisms)

You eat GMO corn, soybeans, canola and cottonseed oil Probably at least weekly 46% of GMOs are grown in US Europe had 12 year moratorium on growing GE foods