Molecular Diagnostics Flashcards
Hybridization and
PCR
are used for what?
They can be used to
- Identify infectious agents: however the sequence of the pathogen must be known.
- Identify a inherited disorder: however the sequence of the gene must be known.
What must we know before performing hybridization and PCR?
The sequence of pathogen and gene
How many pathogens have been sequenced?
The sequence of 5,000 pathogens have been sequenced
Flies and humans share ____ of genes
50%
Hybridization ideology
ssDNA has a high propensity to bind with another strand of DNA/RNA that is complimentary.
When do we use hybridization?
Hybridization is used to detect a particular DNA/RNA sequence in a mixture of other DNA/RNA.
Short, single stranded oligonucleotides (probes) are designed and radioactively labeled and then immobilized on a a membrane (blotting). The goal is for them to behind with a target nucleic acid that has a complimentary sequence.
_______ is a technique that is based on hybridization.
Blotting.
Types of blotting
Southern
Northern
Western
Southern blotting
Both the probe and target nucleic acids are DNA
Northern blotting
Probe is an antibody
Target is RNA
Western blotting
Probe is a ssDNA
Target is protein
How does blotting occur?
Say you have a cup with a long strand of DNA. You want to see if it contains gene A, your gene of interest.
- Take the DNA and cleave it, resulting in smaller pieces.
- Run a gel electrophoresis to seperate the DNA fragments based on size and charge
- Transfer the gel onto a membrane (blot), which will also transfer the fragments.
- Expose the membrane to our radioactive DNA probe, which is the compliment to the gene of interest. If our membrane has our gene of interest, the probe will hybridize with it
- Use a Xray to visualize it.
Purpose of southern blot
Determine which restriction fragments are associated with a gene of interest.
Purpose of northern blot
Measure the [size] and [quantity] of mRNA
Purpose of western blot
Measure the [amount] of protein or antibody.
What is the purpose of Polymerase Chain Reaction (PCR)?
The purpose of PCR is to amplify a sample of DNA to create millions of copies
How is PCR conducted?
- Select a region of dsDNA that you want to amplify
- Heat to separate strands
- Cool to anneal primers
- Add [Taq polymerase] and all 4 dNTPS (deoxynucleotide triphosphates)
- Amount of DNA is now doubled.
How are the primers designed in PCR?
Primers are designed to flank each end of DNA in a 3’–>5’ direction.
What is Taq polymerase?
creates the copy of DNA by extending the primers.
Where is PCR conducted in?
Thermocycler
Advantage of PCR
We only need a small amount of template DNA: there is a 10^9 fold amplification
Disadvantage of PCR
- You need to know the sequence of the flanking DNA for the primers
- errors
- you can amplify contaminated DNA
Why is PCR ran?
PCR is ran so that you can amplify DNA. Many machines require you to have a certain amount of DNA or you may need more than 1 copy to manipulate the DNA.
Thus, you can use it to
- Detect infectious agents
- Detect genetic mutations
1 cycle of PCR makes ____
2 cycle makes ____
3 cycles makes ____
2
4
8
2^n
n=trial
is PCR quantitative?
No. We are making millions of copies
So we have invented a new method: qPCR
If we were asked:
What method would we perform if we were asked to quantitatively measure much how our gene expression changes in cancer?
qPCR
What does qPCR do?
And how is it different than PCR?
qPCR is used to quantify the copy number of a specific gene.
The only difference between PCR and qPCR is that a probe is used which fluoresces only in the presence of the PCR
What is the probe in a qPCR
It is usually a complimentary oligo with a fluorescent tag.
qPCR is used to do what?
- Detect the LEVELS of an infectious agent
2. Determine LEVELS of gene expression
In the graph indicating [fluorescence vs. cycle numbers], what does a low Ct amount mean?
low Ct= high amounts of targeted nucleic acids
What does a high Ct amount mean?
High Ct= lower amounts of targeted nucleic acids.
Thus, more amount of cycle numbers were needed to produce the same amount of target sequence than the one with the low Ct. In other words, it has less of the target sequence that you’re looking for.
_____ is often used to come up with more specific answers
qPCR
What 2 properties of our DNA can we use to use PCR and hybridization to answer specific questions?
- RFLP–> restriction fragment length polymorphism
2. VNTR–> variable number of tandem repeats
RFLP and VNTR can be used for
- Forensics
2. Dianostics such as (prenatal diagnosis, newborn screenings and genetic carriers)
RFLP Theory and How It is Done
Peoples genome differs by 1 in every 1000 BP. Some of these differences are in the recognition sequence for restriction enzymes. Thus, when attacked by a restriction enzyme, different fragments are made.
In RFLP,
- The genomes of 3 individuals (1 from evidence and 2 from suspects) are cleaved with the same set of restriction enzymes.
- Fragments are then loaded onto a agarose gel, producing different patterns.
- DNA is then transferred to a NYLON membrane
- Conduct Southern Blot
a. Add DNA probe and wash off excess.
b. Put in under Xray
The person of interest will have fragments of the same length, because the same restriction enzyme was used.
RFLP is used in
- Forensic analysis,
- DNA fingerprinting,
- paternity testing
- inheritance of a dz
If we wanted to run a RFLP and our sample was limited, what could we do?
Run a PCR before
2 parents want to use their DNA to determine if their daughter will have sickle cell anemia, what test is ran?
RFLP
RFLP can be used to detect mutations
How can RFLP be used to detect for mutations?
When a person has a mutation, often times the site where restriction enzymes act is altered. You can thus under RFLP using the same restriction enzymes to see if your DNA is cleaved how the mutated DNA would be cleaved like.
How can RFLP be used to detect for sickle cell?
In a normal HB gene, the restriction enzyme 3Ddel will cut at three different places.
Patients with sickle cell, however will only have 2 restriction sites.
Knowing this information, you can run a RFLP to detect for mutations.
Variable number of tandem repeats (VNTR) is used for?
VNTR is used to identify diseases and indicate the severity
What is VNTR
Patterns of short tandem repeats (short repeats of the same nucleotide) occur in the genome, but vary in individuals
What can VNTR be used to detect
- Huntingtons
2. Fragile X syndrome
People with Huntingtons have a large number of _____ copies
CAG
We can make a large scale production of recombinant proteins such as
- insulin
- eyrthropoietin
- growth factors
- clotting factors
- vaccines
How do we create insulin?
We need a: plasmid vector and peice of cDNA.
cDNA is inserted in a plasmid vector to produce a recombinant DNA.
Recombinant DNA is then inserted into a bacteria.
Using a fermentation tank, the recombinant DNA will then produce our insulin, which is then extracted from the bacteria.
How is the flu vaccine made?
FDA selects 3 strains of vaccines it thinks may be selected.
Strains are then incubated in a chicken egg and mixed and distributed.
Flu vaccine starts in ______ and vaccinination begins in ______
January
October
The production of antibodies/antigens are becmoing very common.
ANTIBODIES
What do antibodies do?
Antibodies are released from your body to attack foreign aliens.
Abciximab
a monoclonal antibody that inhibits the aggregation of platelets
Basiliximab
a monoclonal antibody that prevents the rejection of a kidney transplant
Cetuximab
a monoclonal antibody that treats colorectal cancer
Infliximab
a monoclonal antibody that treats autoimmune diseases
Retuximab
a monoclonal antibody that treats lymphomas and leukemia
how are monoclonal antibodies made?
- Rat in injected with antigen
- Spleen cells of the rat will begin to produce antibodies.
- Fuse spleen cells with tumor cells, creating hybridomas and causing it to become immortilized.
- Culture in a HAT medium and select for + cells (cells that you want to bind to the epitope of interest.
- Harvest the monoclonal cells
Monoclonal antibodies have been used to try to treat alzheimers. why hasnt this worked?
Researchers are thinking that the researchers are selecting the wrong epitope for the antibody to bind to
Antibodies are also called
B lymphocytes
ELISA stands for what?
Enzyme-linked immunosorbent assay
What does ELISA do?
ELISA will test for the CONCENTRATION of antigens or antibodies in a sample
Sandwhich ELISA
Measuring the amount of antigen in serum
Thus, the well is coated with antibodies
Indirect ELISA
Measure the amount of antibodies in serum
Thus, the well is coated with antigen
In sandwich ELISA, we are testing for the amount of _______ in the sample
antiGIN
How do we run an elisa
- Well is coated with an immobilized antibody
- A sample with antigens is added. If the specific antigen is present, it will bind to the antibody.
- Wash excess antigens
- Add a a second antigen+HRP (Horserasdish peroxidase), which will bind to the specific antigen, if present.
- Wash
- A substrate (tetramethylbenzidine) is added and the enzyme will cause a color change.
What does the color change in an ELISA tell us?
The rate of the color change tells us the amount of antigen/antibody that is present.
What enzyme is used in ELISA to initiate color change?
HRP: Horseradish peroxidase
What substrate is added to an indirect ELISA?
TMB: tetramethylbenzidine
How do we test for HIV?
HIV testing is performed using indirect ELISA.
Wells are coated with HIV antigens.
We load the well with our serum. If we have HIV antibodies, they will bind to the antigen and we will see this.
+ of ELISA
Efficient,
Sensitive
Cheap
- of ELISA
Can produce false positives and false-negatives
SOOOO confirm with western blot
MI can be detected by performing what?
Sandwhich ELISA.
During an MI, we have elevated levels of Tropinin I and Tropinin T (both antigens).
Thus, we perform a sandwhich ELISA to test for levels of antigen (tropinin I and tropinin T)
What do pregnancy tests for
HCG
Is HCG an antibody or antigen?
Antigen
What kind of test is a pregnancy test?
Sandwich ELISA
We are looking for levels of HCG (an antigen) in our urine.
Pregnancy test has 3 sites
- Reaction site (Site where you pee on)
- Test site (site that tells you results)
- Control site (proves test is working)
Reaction site of a pregnancy test
On the reaction site of a pregnancy test:
- Free HCG antibodies are located.
- We pee on the stick and if we have HCG antigens, it will bind.
- migrates to the test site
Test site of pregnancy test
- has immobolized HCG antibodies
- When antigens+ab complex comes in, it will bind to the immobilized HCG antibodies.
- dye will turn colors.
Western blotting allows you to
VISUALIZE your results.
Western blot is also called
immunoblotting
Western blotting is used for what?
to detect levels of proteins
Our proteins are always antigens, so we are ALWAYS taking the antigen
Western blotting test
- Run our sample of proteins through a SDS page, which will separate our proteins (antigens) based on charge and size (smaller will travel faster).
- Transfer to a nitrocellulose membrane
- Add primary AB
- Add a secondary AB with a tag. Tag will turn the fragmented line colors!
Thicker the line, more of the target protein present.
What tests do we do to CONFIRM HIV?
Western blot
In qPCR, a sample that has a higher amount of target DNA would reach the threshold at a earlier/later cycle?
Earlier cycle
lower Ct
An increase in the amount of PCR product depends on what?
the amount of target sequence being amplified.
_______________ are being made into popular drugs.
monoclonal antibodies.
Why are monoclonal antibodies called what they are?
They are specific for a single epitope on a antigen.
