Lecture 3: DNA Techniques Flashcards
Restriction Endonuclease
A bacterial enzyme whose function is to destroy foreign DNA that may have invaded bacterial cells.
Part of the modification-restriction system.
Modification-Restriction System
A specific system in bacteria whose purpose is to distinguish between foreign and endogenous and invading DNA, ultimately resulting in ]the destructing of invading DNA.
Endonucleases and methylases take part.
Methylase
Causes methylation at the specific sequence wherever it occurs on the endogenous DNA.
Restriction Fragment
Many endonucleases have been isolated, which allow for creation of a wide range of restriction fragments.
These fragments have useful purpose in laboratory study of DNA, etc.
How do endonucleases operate?
Cleave double stranded DNA at sequences specific to that endonuclease.
Ecor I
An endonuclease that yields fragments with 5’ overhangs.
Hha I
An endonuclease that yields fragments with 3’ overhangs.
Hae III
An endonuclease that yields fragments with blunt ends.
Gel Electrophoresis
A method in which fragments of DNA can be placed into a gel and sorted according to their size.
Larger fragments move slower as they are pulled from the anode to the cathode.
Pulsed-Field Gel Electrophoresis
A “fancy” technique used to separate very large fragments of DNA. Otherwise similar to GE.
Southern Blot Analysis
A technique use to identify DNA fragments that bear segments of DNA that are under study.
Can directly detect deletions, insertions, and point mutations.
Steps in a Southern Blot Analysis:
- Fragmentation by a known endonuclease.
- Gel electrophoresis to separate fragments.
- Transfer of gel contents to membrane by blotting (denatured into ssDNA).
- Adding ssDNA or ssRNA probe.
- Autoradiography.
Restriction Map
A way to detail the distributions of the many restriction sites of various endonucleases on a given gene.
Hemoglobin S
Sickle cell hemoglobin.
Results from a point mutation.
RFLPs
Restriction fragment length polymorphisms.
Northern Blot
A technique used to detect the size and amount of specific mRNA molecules.
Otherwise, the technique is very similar to a Southern Blot.
Western Blot
This technique is to proteins what Southern blotting to DNA and Northern Blotting is to RNA.
Permits the establishment of the size and amount of specific proteins.
How are Western blots performed?
A mixture of proteins are placed on an SDS-polyacrylamide gel.
Small proteins move more rapidly than larger proteins.
After transfer to a membrane, they are probed and autoradiographed.
What is SDS?
An anionic detergent that denatures proteins and gives them all an essentially uniform negative charge.
What is the primary difference between Southern/Northern blots and a Western blot?
Southern/Northern blots rely on nucleic acid-nucleic acid interactions, where Western blots rely on protein-protein interactions.
What are two techniques used to study protein-DNA interactions?
DNA band shifts and DNase I footprinting assay.
What is the principle behind DNA band shifts?
It is dependent on the fact that a fragment of DNA to which a protein is bound will be retarded in a gel relative to the same DNA fragment without protein bound.
What are DNA band shifts useful for?
Establishing whether particular proteins (such as transcription factors) are present in tissues of interest.
How does a DNase I footprinting assay work?
Protein is added to asymmetrically labeled DNA and the reactions are then treated with a small amount of DNase I which digests DNA for a transient period of time.
Regions of DNA covered by a bound protein will be protected from digestion and will not appear in a band.
PCR
Polymerase Chain Reaction
What are the steps of PCR?
- Denaturation
- Annealing
- Extension
