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 Taq polymerase?
A DNA polymerase purified from a thermophilic organism, Thermus aquaticus, that survives repeated cycles of boiling
What is important about the final PCR products?
The 5’ ends actually are the synthetic primers, which allows for other things to be attached like biotin, avidin or a fluorescent dye (can then use the product as a hybridization probe for a tissue sample using FISH)
-> can use PCR for detection and also to make reagents for other molecular methods
How are insertions and deletions detected by PCR?
By assaying the size of the PCR product, usually with a method called gel electrophoresis
-insertions and deletions alter the bp quantity (size) of the PCR product
-must first know what the deletions or insertions are and make primers to flank them
What is the upper end size for PCR to work?
5k bp
-Anything bigger gets to be too difficult
Where must the primers be in order for them to detect an insertion or deletion?
For an insertion, the primers will be adjacent to the insertion site
For a deletion, the primers beyond the deletion point
-basically to the sides for each
What happens with a chromosomal translocation?
Parts of 2 chromosomes are brought together in the same DNA molecule, which creates a PCR target that is not present in normal cells
-if the sites where the 2 chromosomes break and rejoin can be anticipated, they can be detected by PCR
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’)
When does PCR work for chromosomal translocations?
If the break points occurs in common places that produce clinically relevant rearrangements - must know break sites beforehand
-Often detected with FISH
What is “end-point” PCR?
Finding out if a product is made (can determine size too)
- do 30-35 cycles
What is qPCR?
=quantitative
-requires cycling the temperature while also measuring amount of dsDNA produced
-most often done by adding a fluorescent dye
-more expensive and takes longer to analyze
What measures can qPCR provide?
viral load
amplification of an oncogene
What is not present during qPCR during the denaturation step?
dsDNA
-very little during annealing
What is Cp?
=crossing point
-the threshold amount of signal needed in PCR to be sure a signal is being seen
What is the relationship b/w Cp PCR cycle and the number of target molecules from the start?
An inverse relationship
-> more target molecules in the sample at the start of the process means reaching Cp in fewer cycles
Key relationship b/w cycles and target molecules
Fewer cycles means more targets in the sample
A one-cycle change is a 2-fold difference
How do you calculate the number of copies of target formed knowing the cycle number?
2^cycle number
How is PCR used for RNA?
Similarly to DNA but with an additional first step in which strands of RNA are copied into single-stranded DNA by an RNA-dependent DNA polymerase (reverse transcriptase)
What does RT-PCR mean?
-reverse transcriptase PCR
(do not confuse w/ rtPCR = real time AKA qPCR)
What is cDNA?
=complementary DNA
What primer is needed for RT-PCR and how is it made?
A DNA primer
-use a large oligonucleotide to synthesize a cDNA that is the antiparallel DNA complement of the target RNA molecule
-OR use short, random DNA fragments that will anneal with all RNA molecules in sample to make a cDNA library w/ ssDNA complements of all the RNA present
What happens during the first cycle after the reverse transcriptase step?
The first cycle of extension turns ssDNA into dsDNA so then everything in RT-PCR is the same as any PCR w/ a dsDNA target
Can PCR be done with RNA?
not directly, but RNA can be converted to DNA with reverse transcriptase to use PCR.
So, we can only do PCR w/ DNA
What are SNPs?
single-nucleotide polymorphisms = bases in the genome whose identity varies among people
ex. Most common version of beta globin gene encodes one form of Hb, variant that -> sickle cell disease differs by a single change in a single base (SCD allele is HBB-E6V b/c it changes a glutamic acid residue at position 6 in the HBB gene to valine)
How can we identify SNPs?
- DNA sequencing
- direct hybridization using 2 oligonucleotides that are perfect complements of the 2 sequence variants
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
What are the FISH “break apart” and “fusion” strategies?
Break-apart: expect the probes to co-localize unless translocation separates them
Fusion: expect the probes to be separate unless a translocation brings them together
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
What tests do we need to know what we are looking for?
FISH
PCR
standard DNA sequencing
* must have a probe or primer for these to look for target
What are advantages of microarrays and high throughput DNA sequencing?
Don’t need to know what we’re looking for in advance
-take a broad, unbiased view
What technique do microarrays use?
direct hybridization
How are microarrays done?
- Synthetic oligonucleotides are attached to a solid substrate in a known order
- Annealing/hybridization occurs
- determine the sequence of the target captured based on location of the annealing spot on the array after washing
What is the purpose of knowing the order of oligonucleotides in a substrate?
If we get an hybridization probe to anneal to a spot on the array, we can look up what the probe stuck to and then know what the sequence of the target captured
What are the different names for RNA and DNA-measuring microarrays?
RNA = gene expression arrays
DNA = comparative genomic hybridization arrays
What are gene expression arrays?
- measuring the abundance of many RNA targets simultaneously
What does the abundance of RNA molecules tell us?
Abundance of RNA molecules in a cell tells us the steady-state level of individual transcripts
ex. how high is the expression of an oncogene like CMYC or how low is the expression of a tumor suppressor like PTEN?
Difference b/w traditional and real time PCR
Real time (qPCR) includes sssDNA fluorescent probe. The intensity reflects the quantity of DNA.
Reverse transcription PCR (per Physeo)
amplifying mRNA to cDNA
*cDNA does not contain introns b/c it’s made from mRNA (mature mRNA lacks introns)
-can be combined w/ qPCR to quantify the amount of mRNA in a cell (can be helpful for ex in tx of cancers to determine how much mRNA the cancer is producing) by first converting mRNA to cDNA, which is then quantified using qPCR
What is a western blot?
process that uses antibodies to detect specific proteins after denaturing them (unfolding) and separating them by size
-informs us about abundance and size of individual proteins
What does polyacrylamide gel electrophoresis do?
Separates proteins by size (SDS-PAGE, protein-detergent) complexes migrate from the top of the gel with a negative charge to the bottom with a positive charge. Smaller proteins travel faster, further
How can we detect the amount of p53 in a cell?
Western blot
-levels are normally very low in human cells, but increase after DNA damage (which can be induced w/ X-rays)
Example of RNA (or cDNA library) microarray
Example of signals from microarray abnormal vs normal
What are heat maps?
a representation of data as a map or diagram. The data values are represented as colors
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 is comparative genomic hybridization?
Measuring DNA copy number variation
-microarrays w/ specific sequencing to ID changes in genomic structure like deletions, insertions, or amplifications (regions of copy number variation..CNV)
What is whole genome CGH?
Arrays that represent the entire genome and measure CNV globally
-can reveal multiple aneuploidies
When is direct hybridization used for screening?
to screen patient samples for genetic variation as small as the single nucleotide polymorphisms (SNPs) like the one associated with Sickle Cell Disease
When is FISH helpful for screening?
to ask if chromosomal domains that should be in different parts of the genome have become associated with one another, if regions that should be together have been split apart, or to ask how many copies of a region are found in a cell.
Microarrays vs gene expression arrays
Microarrays allow us to monitor the abundance of as many as a million target sequences simultaneously.
Gene expression arrays tell us the pattern of RNA molecule abundance in a sample, which in turn can tell us which pathological process is occurring
What does CGH tell us?
Comparative genomic hybridization arrays (CGH) tells us the pattern of abundance of DNA molecules across a patient’s genome, detecting copy number variation (CNV) such as deletions, duplications, or aneuploidies
What are different types of DNA and RNA sequencing?
What is helpful about sequencing?
Determining the nucleic acid base sequence can tell us about cellular functions
-can measure gene expression from the RNA pool
–can tell us if gene has a variant sequence to understand clinical presentation
-can measure CNV
What can high throughput sequencing tell us?
the sequence and the abundance of targets
What are chain terminators?
RNA and DNA analogs that lack a 3’ hydroxyl group and can therefore be incorporated into a growing chain, but cannot have more nucleotides added afterwards
What type of screening uses chain terminators?
Sanger sequencing for DNA
When are nucleoside analogs lacking a 3’ hydroxyl used?
As antiviral therapeutics (ie. Gancyclovir) b/c some viruses can activate them to the triphosphate form to be used by DNA polymerase but normal human cells leave them as harmless nucleosides (no phosphate, no triphosphate, not used in replication)
How much DNA sample is needed for Sanger/chain termination sequencing?
100 ng of purified DNA sample
Do we need to know the sequence for Sanger?
Yes (like PCR), need to know the sequence or at least part of the target to extend a single primer in the sequencing reaction
How does Sanger sequencing work?
- get DNA sample
- denature it
- anneal w/ (single) primer
- add DNA polymerase to extend the primer (add all 4 dNTPs: dATP, dTTP, dCTP, dGTP each with diff colored fluorescence plus a small amount of 4 nucleoside analogs lacking the 3’ hydroxyl w/ a modified base to make it fluorescent)
- denature the product
- separate products by capillary electrophoresis (can separate ssDNA molecules that differ by one nucleotide in length)
- Sequence of the template can be read according to the order of dyes as they emerge (= electropherogram)
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 an electropherogram and what can be learned from one?
The results from separating Sanger sequencing products by capillary electrophoresis, shown in different dyes.
- can be inspected for mutations or abnormal calls, like 2 product peaks at the same size, indicating heterogeneity in our input DNA
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
Chain termination sequencing vs high throughput
Chain termination - less expensive and faster . High resolution. Cheap DNA sequencing for fragments ~1000 bp
High throughput (includes microarray) - more information w/ lower reliability (cost can be reduced by combining samples from diff sources)
How is a sequencing panel developed?
- choose a set of targets (a panel of genes commonly mutated in a cancer or the exome (for exon sequencing)
- Array of oligonucleotides is prepared
- Sample RNA, cDNA or genomic DNA is annealed to the array
- non-complementary sequences are washed away
- target molecules eluted
- sequencing libraries are prepared and processed
What is the exome?
the sequence of all the exons in a genome, reflecting the protein-coding portion of a genome. In humans, the exome is about 1.5% of the genome.
What does a sequencing panel provide?
- detects copy number (gene expression)
- detects sequence variation
What are benefits of using sequencing panels?
Combine array-based purification of specific subsets of target molecules with high-throughput sequencing to reduce the cost and informatic requirements
What methods do liquid biopsies use?
High-throughput to sequence and count copy numbers of targets in circulating cell-free DNA
When are liquid biopsies used?
prenatal screening for aneuploidy
dx of many cancers
How does electrophoresis separate macromolecules?
By the ratio of charge to mass
How do macromolecules carry charge?
nucleic acids - negative charge from phosphodiester backbone causes RNA and DNA to be negatively charged
proteins - charge is variable depending on acidic and basic residues and its pH
How does electrophoresis work?
- Apply an electric current to a solution w/ nucleic acids or proteins
- macromolecules will accelerate toward the cathode (+) or anode (-) terminal at a rate that depends on the ratio of their charge to their mass (F=ma)
- We use denaturing detergents that -> similar charge/mass ratios for molecules of different sizes, so we force them to migrate through gels w/ small pores
- smaller molecules migrate faster b/c they fit through the small pores
What is the main interest in doing electrophoresis with nucleic acids?
To determine the distance b/w sites of the PCR primer binding on a target changes (and therefore alters the size of the product - shorter for deletion, longer for insertion)
To determine the sequence of the template in chain-termination reactions by looking at the order of product appearance after separating them by size
What is the monospot test?
RBCs from a sheep or horse are used as the test antigen to search for the presence of target proteins
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
What are western blots used for?
To determine the size (if it has grown larger or is smaller indicating insertion or deletion or post-translational modification like covalent attachment of another protein, like ubiquitin) and abundance of specific proteins in a sample
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
What is SDS-PAGE?
A gel electrophoresis technique that uses SDS to denature the proteins and add negative charges to the proteins to give them all about the same charge/mass ratio and shape
PAGE = polyacrylamide gel electrophoresis. The gel is a matrix of polyacrylamide that allows separation by mass
-most common electrophoresis used for western blots
What can be analyzed by electrophoresis?
RNA
DNA
proteins
What are 2 approaches to detect proteins in a sample without using electrophoresis?
- Use antibodies from a source animal to look for antigens in the sample
- Use purified target proteins to see if the sample contains antibodies against a target
What are 3 stimuli for clonal expansion?
- antigen exposure (norm, tansient)
- chromosomal rearrangements (abnorm)
- genetic mutations (abnorm)
What are 6 ways clonal expansion of B cells can be measured?
- Immunoglobulin light chain expression by flow cytometry
- immunoglobulin light and heavy chain expression by molecular testing
- aberrant antigen expression by flow cytometry or IHC
- genetic testing (pyrosequencing or NGS)
- karyotype analysis
- FISH
