Molecular Techniques DLA Flashcards
What is a restriction endonuclease?
Cut DNA within a segment of DNA were discovered in bacteria
What were class 1 restriction endonuclease?
Recognize specific sequence but cleavage site is non-specific thus not useful for gene manipulation
What are class 2 restriction endonuclease?
- Cut DNA in a precise and reproduction fashion
- Cuts dsDNA within a symmetrical recognition site (normally)
- Hydrolyze phosphate backbone to give a 5’-phosohate and 3’-OH
- Blunt or sticky overhangs a few bp in length
- Enzymes are homodimers that recognize short, symmetric DNA sequences
What are class 3 restriction endonuclease?
Recognition site is nit symmetrical, not useful for gene manipulation
What are exonucleases?
Cut DNA one base pair at a time from the extreme 5’ to 3’ end of a piece of DNA
What are palindromic regions?
When read in 5’ to 3’ direction, the sequence on the “top” strand is identical to that if the “bottom “ strand
Summarize the function of DNA ligase
- DNA joining enzyme called ligase
- Catalyzes the formation of phosphodiester bond
- 5’ phosphate of one nucleotide to the 3’ hydroxyl of the next nucleotide
What is the purpose of DNA ligase?
Restriction enzyme recognizes specific sequence of DNA
- Cleaves phosphodiester bonds of the top strand and bottom strand leaving 5’ overhanging free nucleotides
- These nucleotide overhangs nicknamed “sticky ends” (also called cohesive ends) can re-establish H-bonds
- DNA ligase re-forms the phosphodiester bonds (on both sticky ends and blunt ends)
What are the 4 general types of DNA polymerase used in labs?
- DNA polymerase 1(from E. Coli or T4 phage)
- 5’ to 3’ polymerase, exonuclease activity in both directions
-Used in Nick translation, probe prep, DNA repair & making a blunt end from sticky
- Klenow fragment DNA polymerase
- 5’ to 3’ polymerase & 3’ to 5’ exonuclease
- uses single stranded template
- Used in Sanger sequencing & synthesis of second strand of cDNA(cloning) - Reverse transcriptase (RNA Dependent DNA polymerase)
- 5’ to 3’ polymerase & 3’ to 5’ exonuclease.
- Needs RNA as template - Taq polymerase
5’ to 3’ polymerase activity only
- Isolated from thermos table bacteria found in hot spring
- Used in PCR (requires primers)
- New versions contain proofreading capabilities
Explain what is cloning
The introduction of a foreign DNA molecule into a replicating cell, permitting the cloning or “amplification” of that DNA
-Most commonly, to clone a specific nucleotide sequence of interest
- The reaction involves cutting a fragment of DNA and lighting it into a DNA cloning vector creating a RECOMBINANT DNA MOLECULE, then inserting it into a host cell
- A cloning vector is a stable molecule of DNA which can incorporate a foreign fragment of DNA which can be stably taken up by a host cell and then replicate within the host cell
Experimental conditions favor only one piece of DNA inserted into each vector and only one recombinant molecule per host cell
Fundamental technique in recombinant DNA work
Why are naturally occurring bacterial plasmids become cloning vectors?
- Most commonly used vectors are prokaryotic plasmids
- Can insert up to 10kb of foreign DNA
- Replicate inside the cell so that daughter cells have the plasmid as well
What are the 3 important factors for cloning vectors?
- They must be able to self-replicate in a host cell with an origin of replication (both before and after insertion of foreign DNA)
- Must have a region called a Multiple Cloning Site (MCS)
- A number of unique restriction sites, all within the same region of a vector and these restriction sites must be unique and not present anywhere else in the vector - Must contain a selectable marker
- Typically a gene which confers antibiotic resistance to the host cell
-Sometimes it’s a gene for an enzyme that is nit found in the host cell
What are the functions cations cloning vectors are designed for?
- Replication: to replicate & increase the amount of DNA that you have (high copy number)
- Sequencing- contains universal sequences to use for sequencing primer
- Protein expression-
- Multiple cloning site is found dowtnstream of bacterial gene with ribosome binding site
- Genetic information cloned into this vector is translated into protein by the host cell
- Promoter sequence is found in the vector to use host cell machinery to initiate transcription
- Specific called cDNA and will be discussed
Summarize how cloning is done
- DNA fragments to be cloned are created by digesting DNA with restriction endonucleases
- A vector, which replicates autonomously, is selected to act as a vehicle to transfer the fragment into a host cell
- The fragments are then ligated into the vector
- The vector/fragment hybrid, called a recombinant DNA molecule, is introduced into a host cell
- Recombinant DNA will replicate making many copies if itself AKA —> clones
- When host cell replicates, the daughter cells also contain the recombinant DNA, forming a colony of identical cells
- Recombinant DNA may be recovered from this culture of cells- this DNA may be used for many purposes
Summarize the history of PCR
- In vitro method invented in 1983 by scientist Kary Mullis for rapid production of large amounts of specific DNA sequences
- In 1986adapted by forensic scientist Edward Blake and the FBI to perform forensic testing as only a small amount of template DNA is required as starting material
- Proces is automated and each round of replication doubles the total amount of DNA and takes minutes
- 30 rounds (cycles) May result in millions, billions, trillions of copies of a specific sequence
