22: Recombinant DNA technology Flashcards
what are the characteristics of the family with language impairment?
- Simplified speech
- “poor awareness of appropriate sound patterns”
- Poor articulation
- struggled with complex sequences of tongue movements
- genetic
what is the gene related to speech and language disorder
FOXP2 gene on chromosome 7
What can we do to discover the gene?
- clone to determine sequence
- purify protein to investigate properties
- generate transgenic organism
what is a clone? What is a common cloning bacteria?
copy of the original, Escherichia coli
What are the steps to express FOXP2 in E.coli?
- determine which tissues express FOXP2 (northern blot)
- isolate mRNA
- synthesize cDNA
- PCR FOXP2 from cDNA
- Gel purify FOXP2 PCR product (gel electrophoresis)
- restriction-cut FOXP2 + restriction cut plasmid ligated to recombinant plasmid
- transform recombinant plasmid to E. coli
- verify clones (restriction mapping, DNA sequencing)
- purify FOXP2 from E.coli
What are the types of blotting?
Northern: detect RNA using DNA
Western: detect protein using antibodies
Southern: detect DNA using DNA
What are the steps of northern blotting?
- isolate RNA
- separate RNA by gel electrophoresis
- transfer RNA to membrane
- probe membrane for FOXP2
What are the mechanics of DNA gel electrophoresis?
RNA loaded on to cathode (-) side of agarose gel and it runs to anode (+) side
smaller size = runs faster
How is RNA transferred to a membrane?
stack (gel-nitrocellulose paper-stack of paper towels)
liquid soaks through to paper towel taking the RNA with it, but nucleic acid is stoped by biding tightly to nitrocellulose paper
How is the membrane probed for FOXP2?
synthetic oligonucleotide (part of FOXP2 sequence) is incubated with membrane
wash away unbound probe
radiolabel oligo (probe) to detect bound probe on the membrane
probe is complementary to the FOXP2 gene and is detectable to see which cells express this gene (brain, lung, liver, kidney)
What is complementary DNA (cDNA)?
DNA copy of mRNA that is needed for cloning
How is cDNA formed?
- total mRNA prep
- add poly(T) DNA primer to 5’ new end
- use reverse transcriptase to many cDNA
- RNase H degrades mRNA template and E.coli DNA poly and ligase add and ligate new nucleotides where the mRNA template used to be
transcription can begin/end anywhere on mRNA = mixture of strands - result: mixture of cDNA from all mRNAs in tissue
What is PCR?
polymerase chain rxn
rapid amplification of small DNA amounts
What is necessary for PCR?
template sequence to anneal primers
What are the steps of PCR?
- strands separated at 95ºC strand contains target sequence + excess
- anneal primers 50º-60º to target sequence
- extend primers 72ºC using dNTPs and thermostable DNA polymerase
repeat many times, at 3rd cycle, the exact target FOXP2 cDNA region appears and is amplified and can be purified from the other cDNAs present with gel electrophoresis
What are some applications of PCR?
- Quantitation of target DNA using fluorescent dyes
- DNA fingerprinting
- Site-directed mutagenesis (amplify mismatch)
What are plasmids? What are they useful for?
Bacterial DNA fragments that replicate independently of chromosome
* Useful to introduce foreign genes (e.g., FOXP2)
What are the features of plasmids?
- selectable market (antibiotic resistance gene)
- origin of rep
- multiple cloning site within lacZ operon which has consensus sequence for restriction enzyme cut sites
What are restriction endonuclease?
enzymes that Make double-strand breaks at specific sequences, usually palindromes of 4, 6, or 8 bp (cutting at MCS of plasmid)
What are the 2 types of ds breaks done by restriction endonuclease? How can you make compatible ends between the plasmid and insert gene?
- sticky ends (5’ or 3’ overhang)
- blunt end
- ensure blunt end
- compatible restriction enzymes
- making blunt ends with exonuc
- filling up ease with polymerase
How can you create recombinant DNA?
Cut plasmid and foreign DNA to create compatible ends
Incubate insert with plasmid
Sticky ends anneal, ligase seals backbones
What is transformation?
Process by which chemically made competent bacteria take up exogenous DNA
How are the correct bacteria (insert containing) selected for?
ensure cells with bacteria have antibiotic resistance gene,
Plate cells on agar containing antibiotic
* Cells with plasmid will divide to form colonies (assume each colony developed from a single bacteria)
What is the purpose of restriction mapping? How does it work?
verify clones
Cut plasmid with restriction enzymes, check sizes of resulting fragments: if the size is larger, this means that the MCS contains the insert
How else can clones be verified?
- restriction mapping
- hybridization
- PCR
- sequencing
What is Sanger (dideoxy) DNA sequencing? How is it analyzed?
- Enzymatic DNA synthesis in presence of fluorescently tagged ddNTP terminator
- Four separate reactions, one colour for each base
analysis
- pool reactions and denature strands
- separate by capillary gel electrophoresis
- fluorescent tag identifies each terminal base
- graph shows replaced base and which residue of amino acid It is in
What are the steps to purifying a protein from E. coli?
- Create expression plasmid
- Transform cells with plasmid
3.Grow cell culture, inducing over expression - Lyse cells
- Remove cellular debris
- Chromatography (Size, Charge properties, Affinity to a specific matrix)
- Check purity
What is the Anatomy of a protein expression vector?
ori, RBS, promoter, affinity tags, coding sequence for tag removal, terminator, selection marker
What is an example of purification?
SDS page:
1. cell extract
2. flow through fractions from affinity column
3. obtain purified protein and analyse structure and function
What are Transgenic organisms? What are the types?
Organisms whose genomes have been permanently altered by genetic engineering to see effect in a whole organism
- Gene replacement
- Gene knockout
- Gene addition
How are Mice with altered FOXP2 genes?
CRISPR cas 9
Homozygous knockout
- Developmental delay, motor abnormalities
- Fewer vocalizations upon separation from mother
- Premature death at 3-4 weeks
Heterozygous knockout
- Fewer vocalizations upon separation from mother
- Differences in male courtship “song”
