Recombinant DNA Technology And Biotechnology 11/15 Flashcards
Important achievements leading to modern molecular biology
- restriction endonuclease
- cloning of DNA
- creation of synthetic probes (paternity test)
- PCR
Enzymes that cleave very specific DNA sequences
Restriction endonuclease
What kind of sequences are recognized by restriction endonuclease
- Generally short (4-8 bp)
- palindromes
DNA palindromes
Read the same 5’ to 3’ on both strands
Is this a palindrome?
5’ TCTAGA 3’
3’ AGATCT 5’
Yes
Is this a palindrome?
5’ TCTTCT 3’
3’ AGAAGA 5’
No
What kind of ends to restriction enzymes cleave to leave?
Sticky or blunt ends
Sticky ends
- staggered or cohesive
- allows the 2 nucleotide base pair. Helps restriction enzyme. More favorable
Blunt ends
-no orientation regulation, less favorable. Just a clean straight down cut
What is attached after cleavage done by the restriction enzyme?
The 3’ OH group and the 5’ phosphate are attached after cleavage (important for ligation) for both sticky and blunt ends
How are restriction enzymes named?
Generally for the organisms they were derived from
DNA sequences that can be cleaved by a restriction enzyme
Restriction site.
Restriction enzymes that recognize larger sequences…
Cut less frequently
Restriction enzymes that recognize shorter sequences,
Cut more frequently
Tools to cut, paste, and analyze DNA
Restriction enzymes
Recombinant DNA
Fragments of DNA can be “pasted” together to make hybrid molecules
What kind of ends are better for recombinant DNA?
Sticky ends
Enzyme which creates the phosphodiester bonds
DNA ligase
Inserting a restriction fragment (recombinant DNA) into a cloning vector
DNA cloning
What happens to the vector in host cells?
Can then be replicated
inserting a restriction fragment into a cloning vector, vector replicated in host cells, and DNA now cloned and amplified
Recombinant DNA amplification
Molecules of DNA that can accept fragments of foreign DNA
Vectors
Vector requirements
- must be capable of autonomous replication in the cell
- must have at least one restriction site for foreign DNA insertion
- must carry at last one gene for selection (usually antibiotic resistance)
What is the most common vector
Prokaryotic (bacterial) plasmids
What is the second most common vector
Yeast
Two types of DNA libraries
- genomic DNA libraries
- cDNA libraries
Genomic DNA libraries
- entire genome chopped up with restriction enzymes, cloned in vectors, and used to transform bacteria
- each transformed bacteria containting a plasmid may contain a different segment of the genome
- collection contains ALL SEQUENCES IN THE GENOME, including coding regions as well as introns and other intervening sequences
CDNA library (complimentary DNA)
- cDNA generated using isolated mRNA from a particular cell or tissue type
- mRNA is reverse transcribed (by reverse transcriptase) and the second strand is synthesized using a DNA polymerase
- cDNA is ligated into a vector, used to transform bacteria and 1000s of clones are collected
Ideal cDNA library
Will contain sequences representing all mRNAs present in the cell or tissue types at the time the mRNA was collected
CDNA libraries allow
- allow one to see what genes were being expressed in a particualr cell or tissue type
- only contain mRNA sequences ( no introns, promotes)
