10. Gene Manipulation Flashcards
Gene cloning
Isolation and amplification of a given gene
Recombinant DNA molecule
DNA molecule made by joining 2+ different DNA molecules
Cloning strategy
- Excise and isolate the gene from a genome
(Ligation) - Clone gene into a vector molecule (transformation)
- Introduce into host cell that will take it in and reliably replicate it (extraction)
- Isolate gene from host and retrieve it
Restriction endonucleases
Sequence-specific cleavage of DNA by EcoR1 and protection from cleavage by methylation
Make site-specific cuts in DNA
Saves bacteria from phage
Types of restriction enzymes
Blunt-end
Overhang
Blunt-end cutting enzymes
No leftover nucleotides
Overhang cutting enzymes
Provides cohesive ends for re-ligation
Restriction sites
Nucleotide sequences
Restriction enzymes commonly recognize palindromic sequences
Provide info on location of genes
Restriction analysis
Extract fragment (band)
- Clone into a vector & propagate
- Use tag (probe) to molecularly mark region in genome where it is
Restriction fragment length polymorphism
Restriction fragments can be used as molecular markers that reveal a blueprint of individual genomes
Vectors
Naturally small, transferable, and replicate DNA molecules
Act as template
Plasmids
Vector
Can carry over sequences from different genomes
Phage DNA
Vector
Used as a vehicle to infect
How to clone a gene
- Isolate DNA fragment w/ gene of interest (in vitro)
- Insert into plasmid that is a vector (in vitro)
- Grow in bacteria to amplify (in Vivo)
Formation of a recombinant DNA molecule
- Cut plasmid and DNA with restriction enzyme that will create identical ends (digestion/restriction)
- Incubate the 2 DNA molecules together in presence of DNA ligase (ligation)
- Transform to bacteria to propagate and amplify (transformation)
Inserting gene into recombinant DNA plasmid
- Digestion/restriction
- Ligation
- Transformation
Can be used to clone many DNA fragments
Library
Collection of cloned DNA fragments
PCR
Used to amplify DNA in region of interest between 2 primers
Requires knowing the DNA sequences so primers can be designed
PCR requires:
- Template DNA
- Primers
- DNTPs
- Buffer and Mg2
- DNA polymerase that is het resistant
I.e., taq polymerase
Producing PCR products with sticky ends
DNA sequences introduced to DNA via primers used for PCR
Approach can be used for adding terminal restriction sites that help with cloning PCR product
Bacteria=live factories for DNA amplification
Bacteria used to propagate and amplify DNA (e.g., E. Coli)
Direction of insert does not matter if
Goal of cloning is to simply amplify its sequence
Direction of insertion does matter if
If goal is to express gene under regulation of promoter in vector
Only 1 direction will produce recombinant plasmids that will transcribe sense mRNA
(Reduces efficiency of cloning to 50% of ligation products)
Cloning strategies
- Digestion strategy
2. Ligation strategy
Digestion cloning strategy
Choose correct restriction nuclease to use
Ligation cloning strategy
Select the type of vector and method of ligation
Making selections in cloning
- Selection for plasmid background
2. Selection for presence of an insert
Selection for plasmid background
Ensure that the only surviving bacteria carries a ligation product
Selection for presence of an insert
Identify among surviving clones the recombinant plasmids
Steps to cloning
- Insert
- Digest vector at multi-cloning site
- Digest insert
- Ligation insert + plasmid
- Transform with ligation products
- Select for either plasmid backbone or presence of an insert
Cloning large fragments
Fosmids and BAC’s are cloning vectors that carry large inserts
GDNA
DNA libraries that store and study genomes; comprised of individual clones carrying DNA fragments that in association represent part of the whole genome of organism
CDNA library
Complementary DNA (DNA generated from RNA by process of reverse transcription
How to find clone if interest
- Use DNA or RNA probes
- Hybridization of 2 DNA molecules
- Identify the +’I’ve colony to propagate
