Molecular Biology Techniques Flashcards
What is hybridization?
Annealing of ssDNA probe to complementary ssDNA
- sequencing (primer )
- southern blots
- northern blots
- RFLP analysis
- Allele specific oligonucleotide (ASO probe)
- Western blots (protein)
What do we test for in genomic DNA?
-structural changes to the chromosome such as insertions or deletions of DNA leading to a genetic disease
Mutations in the coding sequence of DNA
- point mutations that change the size of mRNA
- Often affecting the reading frame downstream of the mutation
- Often leading to the introduction of premature stop codon
- point mutations in the promoter region leading to loss of transcription
- point mutations in the exons leading to non-functional protein
What are the main factors for our substrate for a analysis ?
Double stranded DNA is isolated from a cell, digested by restriction endonucleases (chromosomal DNA is big) and separated according to size
Single stranded mRNA is isolated from a cell and separated according to size
Protein is isolated from the cell and separated according to size
Lipids,protein and RNA is precipitated & DNA is extracted from a cell solution
mRNA can be isolated from cell solution using a poly-T tail immobilized on a bead
Protein purification complicated by localization in cytosol vs. membranes & organelles
Describe gel electrophoresis
Agarose is a polysaccharide from seaweed
- Powder is mixed with buffer and melted
- Ethidium Bromide is added to the liquid which is poured onto a mould, “comb” to form wells is placed onto the mold and the agarose solidifies into slab
- Comb is removed and the slab is submerged in a buffer solution
- DNA sample is mixed with sample buffer containing high density liquid, dyes to follow the migration of your sample and ethidium bromide to visualize your DNA fragments under UV light
- Electric current is passed through the gel
- Nucleic acids have a negative charge (phosohate backbone), and they migrate toward the positively charged pole
- Pores between the agarose molecules act like a sieve that separates the molecules by size
- Smaller DNA fragments travel faster
What is the function of hybridization?
Identify DNA of interest
A segment of DNA designed to form complimentary bonds to genomic DNA of interest
- Probes are tagged with radioactivity or fluorescence
- Most of the diagnostic techniques that will be discussed will rely on hybridization to work
Explain southern blot analysis
-Probe is created which is ssDNA that is complementary to the gene region you are looking for
- Probe is tagged with either radioactive isotope or fluorescent marker
- Presence vs absence of DNA region
- Presence vs absence of point mutation (make/break mutations)
- DNA is purified from a cell and then cut with restriction enzymes & separated by gel electrophoresis
- Gel is soaked in an alkaline solution (denature DNA)
- DNA transferred from gel to DNA-binding membrane
- Radioactive prove is hybridized to the membrane
- Excess probe washed
- Membrane exposed to film
- DNA fragments of interest is detected
Summarize how southern blots are done
- Genomic DNA digested with restriction endonucleases
- Fragments are loaded on to a gel for electrophoresis
- Fragments are separated according to size
- Fragments are transferred to a membrane
- Hybridize fragments with radioactively labelled probe
- expose to x-ray film
Explain the use of northern blot
- Northern blot is similar to Southern blot only that the sample contains isolated mRNA molecules, isolated from cells, that are separated by electrophoresis
- transferred to a membrane
- Hybriduzed with a radio labelled probe
- Excess probe is washed off, membrane is dried and exposed to film
- Detects amount and size of the particular mRNA of interest
What are western blots used for ?
To detect and quantify the amount of a particular protein
Does protein have a charge?
Yes, each protein has a unique charge and here we are separating protein only according to size. The protein is denatured by heat and treated with a negatively charged detergent which costs the protein and gives it an overall negative charge
Explain western blot analysis
- Priteins are isolated from cells
- Purified proteins denatured with heat and detergent so that the peptide is coated with negatively charged SDS detergent
- The peptides can also be treated with a reducing agent to break disulfide bonds
- electrophoresis of protein separate according to size
- transfer nylon membrane
- Detection using a label antibody system
- Visualization using chemiluminescence, treated membrane is exposed to x-ray film
- Visualization using chromogenic substrate the pigment forms directly on the membrane
Explain sequencing in the dideoxy /Sanger method
- 4 DNA synthesis reactions set up, each with a bit of one ddNTP and all with template DNA, radio-labeled primer, DNA polymerase, mixture dNTP’s
- DNA synthesis will occur, base pairs incorporated in to growing chain until randomly a ddNTP is incorporated
- Synthesis stops! (No free 3-OH group)
- Millions of reaction proceeds, each reaction stops after random ddNTP is incorporated
- We hope that the reaction is optimized so that each base pair is represented from the template
- Each reaction is separated by polyacrylamide gel electrophoresis and the gel is later exposed to X-ray film
- Label is on the complementary strand
Summarize sequencing automated
- Automated DNA sequencing just like the manual technique but there is a different colored fluorescent ddNTP representing each of the four reactions
- Using one lane on a gel (or a capillary tube), products of each reaction will emit a different color fluorescence when it is excited by light (laser)
- Computer produces a printout of the sequence
Explain RFLP Analysis
One base pair change that affects a restriction site
- Restriction fragment length polymorphism
- Polymirphism is typically defined as a sequence variation that is found in more than 1% or the population and clinically harmless
Single base pair change in the DNA may cause creation or destruction of a restriction site
The clinician must know this DNA variation to do the test
There are two ways to do RFLP…
- Historically by southern blot analysis
- Now we can do RFLP more efficiently by PCR amplifying the DNA region of interest and then digesting the DNA product with the appropriate restriction endonuclease
- Gel electrophoresis allows easy visualization of products
