BIOL SCI 215 Study Guide - Midterm Guide: Apoptosis, Body Plan, Kras

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Recombinant DNA technologies
Molecular cloning
Recombinant DNA technology
Isolate, study, manipulate genes from any organism
Manipulation is genetic engineering
Key to modern molecular genetics, medicine, and biotechnology
Uses
Isolate genes to better study the proteins they encode, understand their function in the
cell
Isolate and study genes that are causing human diseases - screen drugs, design rational
therapies
Manipulate genes to improve crops, produce useful proteins (e.g. insulin), create useful
bacteria for bioremediation
NOT the same as recombinant chromosomes or cloning (e.g., Dolly)
Methods to find gene that expresses fluorescence in jellyfish
1. Genetic screening - mutagenize jellyfish and breed to find recessive mutations causing
lack of fluorescence and then map that gene to find the gene required for fluorescence
2. Isolate all genes, insert in bacteria and see which bacteria glow up
Two different ways to amplify an interesting gene
1. Cloning
2. PCR
Cloning
- Cut up jellyfish genome
- Place one fragment at random each into one plasmid
- Place one plasmid in each bacteria
- Let bacteria multiply
- BUT cannot do “eukaryotic genes → plasmids → bacteria” because bacteria can’t splice
out introns
- SO use cDNA
Reverse transcriptase (from retroviruses)
Can create a DNA molecule from an RNA template
Synthesizes 5’ → 3’
Design a primer called oligo-dT (poly-T’s) that anneals to polyA tail on mRNA’s 3’ end
Then DNA polymerase comes and elongates, the new strand, creating cDNA or
complementary DNA
: a copy of DNA from RNA
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Make cDNA from jellyfish RNA, which will lack introns
Transcription splicing in live jellyfish
Extract jellyfish RNA
Make cDNA “in vitro” (in a test tube)
Use trick to make second DNA strand
Use a plasmid as a vector for delivery. Plasmids can have a high copy number (hundreds per
cell) and are inherited from one generation to another
Each have one ori (Origin of replication)
Transformation: delivering a plasmid to bacteria
Anatomy of a plasmid
MCS (multiple cloning site)
Useful region for cutting and inserting new DNA
Ori
ampR
Antibiotic resistance to kill bacteria that do not take up plasmid
Recombinant DNA overview
1. Cut DNA using restriction enzymes
2. Paste DNA using DNA ligase
3. Transform DNA into bacteria
4. Screen DNA to find desired new molecule
Cut DNA with restriction enzyme
Restriction enzymes are a class of enzymes produced by different species of bacteria
Each restriction enzyme cuts at a specific DNA sequence
Useful as a defense mechanism by bacteria
Useful for biotech for us
E.g. EcoRI (comes from E. coli): cleaves palindromic DNA to get sticky ends
Restriction enzymes cut DNA at specific sequences to leave either “blunt” or “sticky” ends
Sticky ends = overhanging ends
Why doesn’t the cell’s restriction enzymes destroy its own genome?
DNA methylation protects bacterial genomic DNA from being cut
Acts as an immune system
Ligase joins fragments together
Requires compatible sticky ends
Transformation: Bacteria can be treated to allow uptake of foreign DNA
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Shock bacteria using salts or electricity so they can take up plasmids
Only 1 in ~100,000 bacteria cells takes up a plasmid
Each bacterial colony grew from one cell so are genetically identical
The use of antibiotics and resistance genes allows selection of bacterial cells that contain the
plasmid
Transformations are inefficient, which requires the use of a selectable marker such as
AmpR
Screen for the desired product
Often particular cloning strategy requires testing the plasmids growing individual
bacterial colonies for presence of the insert or orientation
One way to check for insertion is with PCR
Gel electrophoresis
Could sequence
To produce jellyfish GFP by expression in E. coli, you should use a vector with a bacterial
promoter because it needs to be recognizable by the bacteria’s RNA polymerase
Plasmid library: each bacterium expresses a different jellyfish protein
We have now isolated a cDNA sequence encoding GFP protein
5/21
Transgenic
Methods
- Electrophoresis - measure lengths of nucleic acids
- Dideoxy sequencing and PCR - you require a primer
Why would you create transgenic animals?
- Give animals/cells a new protein
- Interested in a particular protein and you want to understand the functionality
- Overexpression
- Determine consequence of having excess protein for that gene
- E.g. Gal80
- Misexpression
- Express myoD everywhere in mouse not just in making muscle
- Expressing a gene in new time and place
- Make a labeled version of that protein/tissues to examine using GFP so
that u can track the growth such as during cell divisions
- Create a specific mutation
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