Wk 1 Molecular Detection Flashcards
How do I detect nucleic acids and proteins?
Like detects like:
detect nucleic acids w/ nucleic acids and proteins w/ proteins
List 4 characteristics of nucleic acids that are important for detection
- RNA and DNA hate being single stranded
- Will form proper base-paired structures if possible
- If not possible, will still achieve duplex form, hairpin if necessary
- Single nucleic acid is expert at finding antiparallel complement
Epitopes
Little bits of proteins
What are antibodies?
Proteins that are selected for detection b/c they recognize epitopes
How are antibodies utilized for detection?
A tag is attached to the Ab so we can see whether the epitopes of other proteins are present in a sample
What does antiparallel refer to?
DNA double strands - one strand runs 5’ to 3’ while the paired strand runs 3’ to 5’
- DNA will almost always be antiparallel, even if base pairs don’t match
What does complimentary refer to with DNA?
Base pairing:
G-C
A-T
How does RNA resolve its nucleic acid’s needs to be paired?
It forms pairs internally in hairpin structures
What does the stability of duplex pairs of nucleic acids depend on?
How well the base pairs match when they line w/ each other in antiparallel manner
AKA stability is determined by their antiparallel complementarity
How are nucleic acids detected for tests?
Add a specific nucleic acid sequence to the sample and test whether its compliment is also there
Steps of finding nucleic acid in a sample
- denature all nucleic acids
- put in probe (known nucleic acid sequence) to reannealing = putting 2 separated strands of nucleic acids
- record if probe was able to find its antiparallel complimentary strand
*example w/ Hb and HbS (sickle cell mutattion) - 2 probes:
1. is antiparallel and complimentary to norm Hb gene
2. is antiparallel and complimentary to HbS allele
-stick DNA sample to paper, compare probe colors to determine annealing in sample
What happens when nucleic acids are denatured?
They dislike being single, so will do anything to form duplex molecule, even as hairpin
What happens when nucleic acids are denatured?
- Pairs break apart to unhappy singles
- Singles collide and most lead to unstable pairings that quickly fall apart
- Rapid “zippering” to create paired strands after a slow initial correct contact
What are 3 things we can look for with regard to proteins in a sample?
- Whether or not a particular protein is present
- Whether a protein is modified
- Amount of protein in the sample
How are antibodies used to answer questions about proteins in a sample? 2 steps
- Find antibodies against protein of interest in an animal, maybe rabbit or mouse. It will only bind to protein of interest.
- Conjugate (chemically link) the Ab to something we can detect - fluorescent dye or enzyme that catalyzes pigment formation
Nucleic acids are made of what?
Nucleotides:
DNA: A, C, G, T
RNA: A, C, G, U
What do nucleotides contain?
A phosphate, sugar, and a base, joined to form long chains
What is hybridization?
AKA annealing
-hybridization is the formation of hybrids (duplexes)
-a probe strand is added to a patient sample base, then pairs with a nucleic acid strand in the sample, forms a stable duplex
What do polymerases do?
Enzymes that read the base sequence in the template strand and add material to partial duplexes at the 3’ end to complete them, until there is no more template
What is the core principle of “extension of primer-template junctions by polymerases?”
We can watch nucleotide choices made by polymerases to determine the sequence of bases in a nucleic acid in a patient sample. This allows for RNA and DNA sequencing.
What are the 2 core principles used to detect and analyze DNA and RNA molecules?
- hybridization/annealing
- extension of primer-template junctions by polymerases
What dictates the function of a protein?
How it’s folded
What are proteins?
Long chains of repeating subunits, amino acids.
Fold to form a hydrophobic core w/ hydrophilic or charged residues on the outside
What are antibodies?
Protein complexes formed by the immune system that can recognize specific amino acid combinations in target proteins
Can be used as probes to detect proteins of interest in patient samples
How are antibodies formed for use as probes?
- A source animal is exposed to the protein of interest
- their immune system recognizes it as foreign
- makes antibodies that bind specifically to the target
- We isolate these antibodies and modify them to be used as probes
How is pathogen exposure determined?
- We purify a protein of interest
- expose patient sample to the protein
- determine if patient has antibodies against it already
- If yes, prior exposure is confirmed
What are metabolites?
Substances made or used when the body breaks down food, drugs, chemicals, or its own tissue = process of metabolism
How is measuring metabolites helpful?
Their amounts in cells or extracellular fluids can offer insight to normal and pathological processes
Define small molecule
smaller than the long chains of nucleic acids or proteins
Examples of metabolites
Na+, K+, sugars (ie glucose), pyruvic acid, fatty acids
Sequence of events that determine the characteristics and functions of cells and organs
Sequence of bases in DNA dictates sequence in RNA, which encodes the amino acid order in proteins, which populates the metabolite pools, which dictate the expression levels of genes
-> DNA, RNA, proteins, and metabolites work together
Difference b/w nucleotide and nucleoside
Nucleotide has:
1. a sugar (ribose or deoxyribose, depending on -OH or -H at the 2’ position)
2. a base (adenosine in ATP)
3. phosphate at the 5’ position
Nucleoside has:
1. a sugar
2. a base
NO phosphate
How are carbons in bases of nucleotides distinguished from carbons in the pentose (5 C) sugar ribose?
Pyrimidies C, U, T given numbers 1-6
Purines A, G given numbers 1-9
Pentose sugar ribose given 1’, 2’, 3’, 4’, and 5’
What is polarity in a molecule?
electron density is unevenly distributed throughout the molecule, resulting in regions of partial negative charge and regions of partial positive charge
Are nucleic acids polar or nonpolar?
Polar
What determines polarity in nucleic acids?
The polarity in DNA and RNA is derived from the oxygen and nitrogen atoms in the backbone. Nucleic acids are formed when nucleotides come together through phosphodiester linkages between the 5’ and 3’ carbon atoms.
Where on nucleotides do polymerases add new nucleotides?
To the 3’ end of an existing primer-template
-grow at 3
What are the roles of the 5’ and 3’ ends of nucleotides?
5’ nucleoside triphosphates (NTPs) are the substrates used by polymerases to build nucleic acids
3’ hydroxyl is the nucleophile for the polymerization rxn in which a pyrophosphate is displaced from the incoming NTP and the nucleotide is added to the 3’ end.
*polymerization always proceeds 5’ to 3’, extending the 3’ end
What parts of double-stranded (duplex) nucleic acids are hydrophilic and hydrophobic?
Bases are flat & hydrophobic (buried in core)
Sugars and charged phosphates are hydrophilic (sugar/phosphate backbone faces the outside)
What is dsDNA? ssDNA?
=double stranded DNA
=single stranded DNA
What kinds of duplexes can be formed?
RNA:RNA
RNA:DNA
DNA:DNA
What is a primer-template junction?
= The juxtaposition of a 3’ end with an antiparallel single-stranded template
- the 3’ end is needed to “prime” the extension rxn and the antiparallel strand is the template that will be copied
How are the strands of duplexes held together?
By the hydrophobic interactions b/w the faces of the bases as they stack and the H bonds formed b/w edges of paired bases
-no covalent bonds -> can be heated for bonds to break (denaturing)
Why are some duplex molecules called hybrids?
After denaturation, it is unlikely strands will pair again with their originally paired strands, so they’re called hybrids because they come from 2 different original duplex molecules
-process = hybridization
What questions can PCR be used to answer?
- Is the target molecule present?
- If present, how abundant is the molecule?
How is PCR done?
2 DNA primers are used to make 2 primer-template junctions that face each other
-both extend (at 3’ ends) w/ a DNA polymerase
-w/ 2 primers, the number of target molecule copies doubles
-repeating this for many cycles -> exponential growth of the target copy number -> “chain reaction” of PCR
What is PCR?
=polymerase chain reaction
What does PCR measure?
the presence, structure and abundance of specific nucleic acids in a sample
-how far apart 2 binding sites are in a nucleic acid molecule
What is the typical run of a PCR?
30-35 cycles
takes 1-2 hrs
gives amplification of 2^30 to 2^35
Raising temp to 95 degrees C to denature, lowering to ~55 degrees C to anneal, repeat
What is necessary to develop a PCR assay?
Need to know the sequence of the DNA target in order to synthesize primers
What are the primers for PCR?
synthetic oligonucleotides - usually ~20 bases of ssDNA
Primer 1 (red) has the same sequence as the “top” strand so will anneal to the bottom strand
Primer 2 (magenta) has the same sequence as the bottom strand so it will anneal to the top strand - orientation is 3’ to 5’ left to right
What do the primers in PCR do?
Anneal to their antiparallel complements and form primer-template junctions
What happens during PCR?
- Primers anneal to their antiparallel complements forming primer-template junctions
- DNA polymerase extends these from the 3’ ends of the primers (Primer 1 to bottom, Primer 2 to top) with both 3’ ends pointed into target
- exponential amplification (chain reaction) of the target
Why is Primer 2 confusing?
Students often get the sequence of “Primer 2” wrong on exams because they treat it as if it were derived in some different way; “Primer 1” is easy to read from the sequence as written, but “Primer 2” is shown 3’ to 5’ in the text above, but then it is written down 5’ to 3’ in exams. If you want more explanation of this point, this is covered in the short video (<2 minutes) linked below as well.
Steps to creating a PCR primer if given a 5’ to 3’ sequence
- write in antiparallel 3’ to 5’ sequence
- Pick the two primers, one top, one bottom
- Write them both as 5’ to 3’
- Remember they are written 5’ to 3’
What is the rate of annealing reaction driven by?
Concentration
So, since the concentration of the synthetic primers is greater that the [] of solitary target molecules, annealing to primers is faster
What is a chromosomal translocation?
2 double strand breaks in 2 different chromosomes (1 in each)
The breaks then connect to the wrong chromosome at its break (5’ to 3’)
What is the process of FISH?
- Put cells or tissues on slide
- denature the nucleic acids in situ (in place, retaining the structural forms as when alive)
- hybridize using a nucleic acid probe that’s been coupled with a fluorescent dye so labeled probe is annealed to cellular DNA
- DNA usually counterstained to look blue to show nuclei (interphase) or condensed chromosomes (metaphase)
- Look in microscope to determine where the probes annealed
What are 4 basic questions we can answer with FISH and what are they testing?
- Is this sequence present? Testing for deletions
- Is this sequence present the appropriate number of times? Testing for duplication or amplification
- Is this locus intact? Testing for translocations
- Have 2 separate loci fused? Testing for translocations
When is the “break apart” strategy used in FISH?
Useful if a single oncogenic partner like ALK has multiple fusion partners b/c it will always get disrupted by translocations but we don’t know (or care) what the partner is each time
When is the “fusion” strategy used in FISH?
Used if translocation usually involves the same 2 regions coming together like in the t(8;14) translocation that leads to Burkitt lymphoma
What are FISH “counting” methods?
Add a probe for a centromere as a control to confirm we get 2 spots for something that should be there twice. This increases our confidence that getting 1 spot or more than 2 really means something
Example of RNA (or cDNA library) microarray
Example of signals from microarray abnormal vs normal
How are heat maps helpful with a microarray?
They can have hierarchical clustering to help sort the results from many patients into groups
-higher than norm expression might reveal an oncogene
-too little expression might reveal a tumor suppressor
-cancers can do multiple inappropriate things, can compare expression from 100 aberrantly expressed genes from 100 tumors, about 1 M data points
- similar abnormal patterns from diff patients can be displayed together
What do we learn from Sanger sequencing?
- DNA molecule size
- the color of dye tells us what nucleotide was last added
- -> the sequence of the template
- -> can infer the sequence of the target DNA
Example of Sanger sequencing
Target DNA molecule at the top
What is high throughput sequencing?
-various versions
-common characteristics: 1. DNA from sample is fragmented and amplified by PCR w/ primers that have common 5’ sequences (“bar codes”) and random 3’ sequences that can anneal to and amplify any target.
2. the bar codes attach the “libraries” to a solid surface
3. another common sequence is ligated to end distal to the surface and is used to amplify the captured fragment and locally immobilize it
4. results in “spots” on the surface w/ hundreds of target copies
5. Surface has millions of spots w/ different DNA sequences but a common primer-binding site that can make a primer-template junction for DNA polymerase to extend
6. incorporation of a nucleotide (like dATP) -> a flash of light or change in conductivity that’s observable
What are “rapid” tests looking for?
The presence of specific proteins using antibodies to see if they bind to the target protein in a sample
ex tests for SARS-CoV-2, rapid strep and home pregnancy tests
How is a western blot done?
- SDS-PAGE
- Proteins are transferred to surface that immobilizes the proteins
- Stain w/ antibodies against specific target proteins to determine the abundance and size of target protein
