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
Explain diagnosis of a patient with suspected sickle cell anemia by RFLP- southern blot
Sickle cell mutation in the beta-globin gene destroys a restriction site Dde1 (notice the other 2 naturally occurring Dde1 sites)
- Patient genomic DNA is digested with that specific restriction enzyme and all Dde1 restriction sites will be cut
- Fragments are run on agarose gel, then transferred to nitrocellulose membrane, hybridization of membrane
- radioactive probe is created to bind the region of interest
- Membrane exposed to film and the pattern is interpreted for diagnosis
Explain RFLP - PCR Analysis - single base pair change
Diagnosis of Maple Syrup Urine disease
Common mutation Y393N which destroys a specific restriction site (Scal)
-Autosomal recessive disease, thus test will reveal carrier status
First: PCR amplify a 186 bp fragment from the MSUD gene
Next: Digest with the appropriate (this test Scal) restriction enzyme and separate DNA fragments according to size by electrophoresis
-visualize DNA fragments using ethidium bromide and UV lights
What are the characteristics of ASO probes?
- Probes are usually 15-21 bases long and very sensitive
- Complementary to DNA of interest
- Used to detect Polymorphisms or common genetic mutations
- CFTR
- Sickle cell
- many others
Contrast the DNA from a normal person and a patient with sickle cell
Patient with sickle cell disease- portion of the gene for the Bs-chain of hemoglobin S
-DNA codes for valine instead of glutamate in the sixth position of B-globin
Oligonucleotide probe hybridizes with a DNA fragment from the gene fir the B-chain of Hb S
DNA from a normal individual- oligonucleotide probe fails to hybridize with the DNA fragment from the gene for the B chain of Hb A
Compare data for PDP1 mutation: sequencing, RFLP & Western
Sequencing- A restriction site created by point mutation in the pyruvate dehydrogenase phosphatase gene which also creates premature stop codon on the mRNA
RFLP- PCR 1084 bp fragment
Normal allele, the 1084 bp fragment should only have one Vspl site (restriction site )
after digestion, two fragments 733 and 347 bp
Mutant allele, the 1084 bp fragment has two Vspl sites
After digestion, 3 fragments 229, 434 & 347 bp
A restriction site created by point mutation which is also creates premature stop codon. mRNA undergoes nonsense nonsense mediated decay, So NO PROTEIN is detected
How to decide when to use ASO or RFLP?
- if the example shows a change in a restriction site, the test will likely be RFLP
- If the example shows a spot, (dot) it us likely an ASO test
- If the example shows allele specific amplification, it is likely ASO PCR
How do we know if it’s a familial mutation ?
-investigation of familial mutation may have a research test, often RFLP which doesn’t need as much optimization as ASO
What to do if you encounter a novel gene mutation as opposed to a known mutation?
Novel mutation requires identification by sequencing
What to do if you have a small sample that presented a mutation?
If small, (ie prenatal sample or forensic sample), you will need to amplify
What to do if you encounter a common mutation ?
These often have rapid diagnostic tests available in many labs, like ASO or RFLP
What structure (protein, mRNA, DNA) would change if mutation caused a premature stop codon?
Protein (ribosome only recognizes stop codon)
Which structure (DNA, RNA, protein) would change if mutation caused splice site mutation?
RNA and protein
Which structure(protein, RNA, DNA) would change if there was a mutation in the promoter?
RNA (RNA polymerase wouldn’t be able to bind and make RNA)
Which structure RNA, DNA, protein ) would change if there was a deletion of exon 2 & intron 2?
DNA, RNA & intron
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