BIOL 325 NUCLEIC ACID AMPLIFICATION STUDY GUIDE

what is polymerase chain reaction (PCR)?

• when a polymerase is used to amplify a DNA target so there is more to manipulate

• commonly called DNA amplification

• start with a small amount of DNA from a sample and every cycle it is doubled by PCR (2^n)

list reasons why a clinical or research lab might perform PCR (written response question)

A clinical or research lab might perform PCR for cloning to insert the cloned DNA into a vector of interest. A clinical or research lab also will perform PCR to detect disease such as COVID or HPV. A clinical or research lab might also perform PCR to detect gene expression with qPCR.

what are PCR primers? (written response question)

PCR primers are single-stranded DNA fragments that must be no shorter than 18 but not bigger than 30 base pairs. PCR primers must be complementary to the sequences that are flanking the region to be amplified. PCR primers determine the specificity of the PCR region.

what do the distance in primer binding sites determine?

determines the size of the PCR product

how should primers be designed?

• primers should be target specific and computers will do this

• primers should avoid primer dimers or interstrand/intrastate homologies

• the melting temperature of the forward primer must be complementary to the melting temperature of the reverse primer

  • important to know the temp to determine where to set up the reaction temp and the primers need to have the same annealing temperatures

• GC content must be 20-80%

• product size must be 100-700 base pairs long

what PCR is used to determine annealing temperature?

gradient PCR

what are the five primer design programs and which one is the best used?

• Generous

• EVERYprimer vendor

• DNAStar

• Idtndna

• NCBI primer blast

  • best program and gets everything first

how do you perform PCR?

• assemble a reaction mix containing all the necessary components for DNA synthesis

  • can use a master mix which already has everything in it which is optimized for clinical applications

  • or do it by hand but there can be mistakes and failures

• subject the reaction mix to an amplification program

• analyze the product of PCR reaction (amplicon)

what is in the master mix or in a standard PCR mix?

• needs to have each primer

• dATP, dCTP, dGTP, dTTP which are all DNA nucleotides

• buffer to keep pH stable like Tris

• salts which are optimized for primer hybridization and are required by the polymerase like KCl or MgCl2

  • salts depend on what polymerase is being used

• the polymerase

  • when the certain polymerase is bought, the buffer, temp, and salt are optimized for it

• 10² - 10^5 copies of template

• 50 ul reaction volume

explain the three basic steps of PCR (written response question)

• the first step of PCR is denaturation where the DNA double helix or anything that is double stranded to single-stranded and is done at very hot temp like 95 for about 20 seconds

• the second step of PCR is annealing where primers are annealed to the target of interest. two primers need to be designed which are forward primers that anneals to the bottom strand complementary in the 5’ to 3’ direction to a 3’ to 5’ strand. the reverse primer is designed to anneal to the top strand complementary and in the 3’ to 5’ direction to a 5’ to 3’ strand. these two primers MUST have the same annealing temperature to optimally work. done at the coolest temperature of the reaction which is around 55 for around 20 seconds.

• the third step of PCR is extension where the polymerase extends the fragment by reading the template and throwing in the right complementary template until it falls off or the time runs out. the temperature of the reaction depends on the polymerase used but it is done around 70-75 for around 30 seconds.

• the final product of PCR is an amplicon where the number of amplicons depend on the number of PCR cycles.

what is a thermo cycler?

• a machine that changes temperature in a block or chamber holding the samples

• PCR requires repeated temperature changes and the thermo cycler does this

what are the thermostable polymerases and what is its use?

• used to withstand the repeated high temperatures

• Taq = thermos aquaticus and is the most commonly used and first polymerase because it works well and is cheap

• Tth = T. thermophilus and is used to do reverse transcriptase

• KOD = T. kodakaranesis

  • is a 3’ to 5’ exonuclease

  • will create blunt PCR product because the polymerase will chew up the 3’ ends so this is used for cloning

• long-range DNA polymerase which is used to make a long fragment

• Pfu = pyrococcus furiosus

  • has a high fidelity so it does not make any mistakes

  • fused to DNA binding protein for processivity

• hot-start DNA polymerase

  • heat is required to activate this polymerase and it is usually Taq or Pfu

how do you interpret PCR results?

• the PCR product should be a size you have expected because you know the primers that are used

  • if there are bands of different sizes that means something went wrong/mispriming

  • misprimes may occur due to non-specific hybridization of primers

• there should be NO primer dimmers which may occur due to hybridization of primers during the annealing step

  • should be avoided and the computer needs to design the primers so that they do not like each other so they do not hybridize during the annealing step

  • a decent size primer band means that the primers used aren’t good

  • if the primers bind to each other then they won’t be able to find the target of interest

• no product should be present in the template blank

how do you avoid misprimes?

• use proper annealing temperature and a gradient machine can be used to find a better annealing temperature

  • too cool/low temp means the primers can bind anywhere

• design the primers carefully

• adjust the monovalent cation concentration/change the salts

• use hot-start

  • prepare the polymerase on ice so it is does not react with anything and place in a preheated cycler

  • use a sequestered enzyme that requires heat (initially 90) to activate

    • platinum Taq

    • AmpliTaq Gold

    • hot starTaq

how do you clean up the product at the end of PCR?

• use gel elution where the band fragment is cut out and isolated by a kit

  • kit could be solid phase isolation of PCR product

  • use an exonuclease to remove the nucleotides and primers prior to sequencing

  • precipitate the DNA

THE PRODUCT AT THE END OF PCR SHOULD NOT HAVE ANY REACTION PRODUCTS LIKE PRIMER DIMERS, MISPRIMES, OR OTHER DNA

what are the negative control components and positive control components?

negative control = shouldn’t work/nothing should pop up

• there should be NO template DNA

• some kits have unrelated target control meaning that the kit should have a negative result when another sequence is present

• contamination control

  • you can contaminate the PCR product and so can working under the same hood

positive control = means the procedure and machine are working

• previous successful sample or one that comes from the kit

• if positive control works but the sample doesn’t then the sample is truly negative

what is optional amplification control?

• target and primer that are unrelated/specific

  • should work TOGETHER

• can be performed in same tube as test sample

• true negative for amplification instead of just amplification failure

  • if sample doesn’t t work but amplification control worked means truly negative

what can be done to prevent contamination in labs performing PCR? (written response question)

Working in an area that is just used for PCR can be done to prevent contamination. The hood must be air-locked and have positive air flow so that any of the outside contaminants do not get in. The PCR hood should be treated with UV to damage any nucleic acid contaminants. Adding dUTP and UNG will chew up any double-stranded DNA and should be done before PCR. Psoralen and more UV should be added post-PCR to destroy any DNA. For surface decontamination, 10% bleach can be used.

what would happen if the primer bound to more than one site?

the amplicon size would be different

is it possible to do PCR without controls?

yes but you wouldn’t know the accuracy of the results; could get false negatives or positives

how can you get results from PCR that has no sample added?

through contaminations like from yourself or other samples

what are the 8 PCR modifications/assays?

1. nested PCR

2. multiplex PCR

3. sequence-specific PCR

4. reverse-transcriptase PCR (RT-PCR)

5. real-time PCR; quantitative (qPCR)

6. whole-genome amplification

   1. AP-PCR, RAPD PCR

7. gradient PCR, Touchdown PCR

8. unamplified target PCR

what is nested PCR?

• when two primer sets are used in one area to amplify a low sample load

• increases sensitivity

• the first set of primer is designed to put on the outside of the target sequence

• the second set of primer is designed to put on the inside of the target sequence to amplify the target again

• when there is not a lot of sample, there is a lot of non-specific binding and in a denatured sample, there will be some primer binding to the targeted sequence but there will also be non-specific binding which might be in the same region or chromosome

what is semi-nested PCR?

when the second primer set includes the same primer from reaction one and the second set still binds to the amplified sequence from the first reaction

how was nested PCR used to detect parasites?

• low parasite loads and atypical lesions can be diagnosed by nested PCR with higher accuracy and sensitivity

• when taking a sample from a lesion, there is not much DNA so nested PCR is used to amplify the sample and can be seen clearer on a gel

what are the advantages of multiplex PCR? (written response)

one advantage of multiplex PCR is that it can process many samples at once. this can be applied when trying to identify a pathogen that a certain individual has by trying to identify many pathogens. another advantage of multiplex PCR is it does not need much sample to be able to identify different genes. another advantage of multiplex PCR is that it cost-effective since many samples can be ran at once.

what is multiplex PCR?

• uses multiple primers to detect duplication or deletion in large pieces of DNA

  • multiple primers in one reaction to pick up a lot of different genes

• most commonly used in forensics labs and to type a pathogen if you don’t know for sure what the pathogen is

  • pathogen typing and forensic ID kits prime several polymorphic regions in same PCR reaction

• in the primer design, the primer amplifies the big fragments and the space between the primers aid in amplifying different size fragments because the gap can vary

• one genital swab or lung wash can be tested for many pathogens in one reaction

how can multiplex PCR be used in the real-world?

• was able to type the multiple types of clostridium botulinum which makes one of the worst toxins known to man

• one primer set amplifies a big region that they all have in common and a second primer set overlaps the area that varies

  • can pick up 5 different types of clostridium because of the multiplexing assay

what is a sequence-specific PCR?

use primers in PCR to sequence DNA or amplify something you know the sequence of and design primers for it

what is reverse transcriptase PCR (RT-PCR)?

• use reverse transcriptase to turn mRNA to cDNA

  • prime the mRNA by making one that is all T’s to bind to the mRNA polyA tail or use random hexamers

• if you do not know what is being amplified, use random hexamers which will amplify anything which will make more cDNA to start experimenting on

  • random hexamers are a bunch of primers that are 6 NT’s long where every sequence is represented and will hybridize to anything in the test tube

• new primer and polymerase to amplify cDNA

• measures RNA expression, detect rRNA, analyze long introns, detect RNA based virus

what is the COBAS amplicor analyzer?

• instrument that is set well to ramp quickly at one temperature to another

• kits are sold that are optimized for the analyzer to get rid of unknowns

• samples are amplified and products detected automatically after the PCR product

• used for infectious disease

  • HIV, HCV, HBV, CMV, Chlamydia, Neisseria, Mycobacterium tuberculosis

what is the aptima assay for HIV?

• gives viral load output/how much HIV a person has which is crucial for determining treatment for an individual with HIV

STEPS FOR APTIMA ASSAY (basically nested PCR)

1. RNA extraction (1-2 hrs)

2. first round of target enrichment (1.5 hrs)

3. second round of step-up PCR (1.5 hrs)

• hybridize amplicons to HIV specific oligonucleotide probes

  • probes are detected by colorimetric determination

what are the advantages of quantitative (real-time) PCR? (written response question)

One advantage of qPCR is that you do not have to wait to see how much amplicon is being produced because every cycle, the amplicons can be seen in real time. Another advantage of qPCR is that it is faster than the other PCR’s and money can be saved compared to southern blotting to identify samples. A third advantage is that measurements taken during the exponential phase can detect the amount of template at the start of qPCR.

what is real time or quantitative PCR (qPCR)?

• used to quantify the amount of starting sample

  • know EXACTLY how much RNA or cDNA or DNA

  • include some fluorescent recorder in the experiment so every single PCR cycle can be looked at (fluorescent recorder)

• can look at viral load, tumor load, and the copy number of genes

• has three phases: log, linear, stationary with a lag phase

• length of lag phase is inversely related to concetration of starting template

  • there will be less starting sample as PCR cycle goes on

• there will be a standard curve to compare your sample to to figure where the cycle number is & how much DNA or RNA it corresponds to

what is threshold level?

• when you first see the fluorescent signal

  • determined by comparing the signal to a background

  • there will always be a background of fluorescence

• need signal to get significantly over the background to be able to trust data

• the threshold level needs to be at a cycle level where background is much lower than the signal to analuze the data

• the instrument is told to capulate the threshold level

what is threshold cycle (ct cycle) ?

• where it exponentially grows in the chart/starts making more product/fluorescence crosses the threshold fluorescence level

  • input is inversely proportional to “threshold” cycle

what are the two DNA-specific dyes?

• ethidium bromide

• sybr green

what are the two hybridization probes and their examples?

• cleavage-based

  • TaqMan

• displaceable

  • molecular beacon, FRET

after reaction completes, what is there no need to do for primer-incorporated probes?

no need to detect products

what is SybrGreen for qPCR?

• SybrGreen intercalates with the double-stranded DNA and ONLY works with double-stranded things

• after PCR, primer and DNA are both amplified into double-stranded targets and when the SybrGreen intercalates, it fluoresces

  • the amount of fluorescence that you can see is indicative of how much product/amplicon is created

• SybrGreen is NOT specific but affordable

what is a melt curve for SybrGreen in qPCR?

• analyzes the amplicons after qPCR

• add more and more heat and if a peak is seen in the chart at a cool temperature (70 C) there are primer dimers

• you want to see peaks over 80 C which are longer annealing products that are specific to the PCR product

• ALWAYS do the melt curve to make sure the target is being amplified but it is still unknown if it IS your target

  • just know that it is bigger than the PCR product than the primer dimer

what is in the devices to detect fluorescence for DNA-specific dyes in qPCR?

• all devices will tend to need some type of primer system and some fluorescent tag

• the devices will all have an excitation system that will filter out one particular wavelength of light that your system uses

  • will pick the wavelength specific to your probe and shine it through the sample

• all devices will also have an emission filter system that reads out of the sample; what you emit is what comes out

  • what is emitted will then be filtered to be the color of light that is specific to the target of interest

• DNA-specific dyes bind and fluorescence double-stranded DNA nonspecifically

• hybridization probes only bind and fluorescence the intended PCR product

how was multiplex qPCR used in the real world in class?

• was used with SybrGreen for the detection of three colistin-resistance genes from cultured bacteria, feces, and environmental samples

  • looked for three different templates in one person’s sample

  • colistin = is a common antibiotic that is developing a lot of resistance and is one of the last potential antibiotics for some GI disorders that are due to bacteria

  • able to look at three different resistance genes to this antibiotic by multiplexing

    • used different fluorophores that slightly emit or excite differently so they emit different color reads for different targets

what is FRET qPCR and what are the steps for FRET qPCR? (fluorescent probe)

• FRET = fluorescence resonance energy transfer

• step 1 = primers amplify to make a lot of amplicons but it is also probed with oligonucleotides

  • the probes purpose is not to amplify it but to give it a reporter system that is more sensitive than SybrGreen

• step 2 = one of the probes has something that can excite nearby neighbors and the other probe accepts the excitement allowing it to fluoresce

• FRET qPCR needs a system that measures the level of fluorescence excitement

• since the two oligonucleotides are specific to the target that have a highly specific assay and know that it is the target of interest

• not expensive

what is TaqMan? (fluorescent probe)

• an exonuclease for Taq

• a little more expensive

• it is proprietary meaning that if you use TaqMan, you need to buy all the instruments and primers specific to TaqMan

what are the steps for TaqMan?

• step 1 = primers amplify the target of interest and a probe that is specific to target of interest is used

  • probe that is bound to target has a fluorophore and a quencher which silences the probe if they are nearby

• step 2 = when PCR rxn occurs and the Taq enzyme creates the second strand, the Taq will chew off the slight overhang that is holding the fluorophore as it goes down the strand when it bumps into it

  • fluorophore is released meaning it fluoresces

  • reporter detected from quencher is detectable by laser

what is molecular beacon for qPCR? (fluorescent probe)

• the probe is in a hair pin/stem-loop formation where it has the fluorophore and quencher

• has the same idea as TaqMan

• when the molecular beacon is not hybridized to the target, it quenches the fluorophore BUT when it hybridizes to the target, the fluorophore is far enough from the quencher where it no longer is silenced and fluoresces

  • does NOT use the same quencher as TaqMan

what happens in Probe-based qPCRs?

• the reagents generate a fluorescent signal that can be quantified to reflect the starting amount of template at each cycle of PCR

what are the similarities of TaqMan and Molecular Beacon?

• The amount of fluorescence increases as the PCR product accumulates/adds up

  • the degree of fluorescence is equivalent to the amount of amplicon you have

• both use sequence-specific primers first then also use sequence-specific oligonucleotides (probe) and the probes have a fluorophore and a quencher

what are the differences between TaqMan and Molecular Beacon?

• in TaqMan, there are overhangs that get chopped off by Taq to release fluorophore

• in Molecular beacons, there is a stem-loop formation and when it hybridizes with the target, it separates the probes far enough to fluoresce

how were the fluorescent probes used in diagnosing leprosy?

• took 6mm skin biopsies by punching the skin and extracting/isolating the DNA

• use a primer and probe system for three genes: one for M. leprae, one for the rRNA from M. leprae, and an internal reference as a control

• have three primer and probe systems in the same time and each dot on the graph represented each patient and was able to amplify and detect all three of the genes in each patient sample

• the earlier of the production of amplicon is picked up meaning there is a lot of target in the sample

  • if it takes past 30 cycles to see anything, there is most likely no target or nonspecific binding

how were fluorescent probes used in the dogs?

• used qPCR to see Leishmania on different breeds of dogs with natural clinical leishmaniosis

• used a primer-probe system to detect Leishmania (high pure PCR template prep kit)

how were fluorescent probes used to see salmonella?

• used a primer-probe system for salmonella which determined the level and species of salmonella in foods that cross the border

• TaqMan was used for this

• number of cases in salmonella in foods that crosses the border has dropped

  • alert vs border rejection has gone up as the years went on due to being tested as soon as it crosses the border

what are the choices for qPCR platforms?

• SybrGreen = nonspecific but easy

• FRET = uses two probes

• Scorpions

• TaqMan

• Molecular beacons

machines are expensive so can only do one or the other not both

what machine do qPCR platforms utilize?

a thermocycler that can adjust temperatures quickly to amplify the target with fluorescent detection and a specialized software

explain how signal-based amplification methods compare to probe-based methods (written response question)

signal-based amplification methods amplify the signal in probes to detect samples with low volume. signal-based amplification does not increase the number of templates. probe-based methods specifically bind to the target sequence and detect that specific sequence. probe-based methods are used to measure the amount of target and to ensure that it IS the target.

why might a whole-genome amplification method by performed? (written response question)

a whole-genome amplification may be performed when a technician does not know what the genome is which can aid in tracing the source of a disease. whole-genome amplification may also be performed for single-cell analysis by amplifying the single cell. another reason why the whole genome amplification method may be performed is to compare genomes to a database full of organisms after amplifying gDNA.

what is the whole genome amplification, arbitrary primed PCR (AP-PCR)?

• can amplify a genome without knowing what you have

  • does not need sequence-specific primers as random hexamers (6 nucleotides long) are used to amplify everything in a test tube

  • random short primers (hexamers) amplifying polymorphic DNA (RAPD)

• has low stringency meaning that there will be a lot of nonspecific binding to the probe

• used to trace disease sources for epidemiology

what is the process for arbitrary primed PCR?

• gDNA is isolated and amplified using degenerative primers

• the first cycle of arbitrary primed PCR creates a bunch of fragments that will get amplified in the second round by the same hexamers or a second set of hexamers

  • a different hexamer in the second cycle can hit and prime off of the same original first hexamer

• the different fragments are sequences in one instrument and will end up with a bunch of sequence information

  • a computer system will recognize how many times a sequence pops up and eventually anneal them together to come up with an organism which can be compared to a database full of them

how was RAPD/AP-PCR applied in the real world?

• used to profile B. cereus and find different strains in vended hot drinks

  • B. cereus is a spore farmer and can survive in temperatures used during vending and the dry, moist humid conditions

• found that 13% of the organisms did not share a RAPD profile

• found B. cereus in the mixing bowl and dispense point of the vending machines

what is gradient PCR? do you need to change the denaturing and extension temp?

• used when you have no idea what the annealing temp is, when primers need different temps, and to determine BEST annealing temperature of primers

  • annealing temp is similar to the melting temp so around the melting temp is where the annealing temperature should be only to allow the primer to bind to the target

• device will put a warm annealing temp first then will get cooler and cooler

• do not need to change denaturing temp because it is always hot at 95 C and do not need to change extension temp because Taq loves 72 C

what do low and high temperature look like in gel electrophoresis in gradient PCR?

• low temp = visible top band

• high temp = visible bottom band

what is touchdown PCR?

• PCR is always run at different temperatures and starts at a higher temp than the optimal melting temperature of primer to force specific annealing for different targets

• the sample is denatured for longer then is dropped down to the annealing temperature then warmed back up to the temp that Taq polymerase uses

  • subsequent 10 cycles reduce anneal cycle then the next 25 cycles are at the optimum annealing temp

  • the temperature is changed every cycle by 1

• touchdown PCR hopefully eliminates nonspecific priming because only specific targets must be hit and amplified enough to be the targets that PCR uses

how come not every primer needs to go through touchdown PCR? when should primers be used for touchdown PCR?

• some primers are good at finding targets

• if primers could potentially cross top to other sequences because of one nucleotide difference between another important sequence in a genome then touchdown PCR is used

how is touchdown PCR used for multiplexing? what is its real world application?

• touchdown is used to adhere to each primer set

  • in multiplexing, you are looking for different targets in one tube so different primer sets are used in one tube

• used to look for antibiotic resistance genes like mecA, bla_SHV, bla_CTX-m, bla_TEM, bla_OXA

  • initial temp is 66 then dropped every cycle by 0.5 C for 20 cycles

what do you do to troubleshoot?

if product is too long

• decrease annealing time and increase annealing temp

  • longer = more nonspecific timing

• decrease extension time or temp

• change monovalent or divalent cations to change nonspecific priming

• use less primer, template, enzyme, check primer for repetitive sequence

do the opposite if the product is too short

ask google

name a technique that does not require an amplified template for detection (written response question)

emulsion PCR is a technique that does not require an amplified template for detection. emulsion PCR is when DNA is chopped up with restriction enzymes and has common primers and ligated oligonucleotides on the ends of each fragment. A wagon wheel formation is sequenced when the chopped-up DNA fragments are emulsified and each drop is unique. emulsion PCR generates single-stranded DNA for high throughput screening and is used to haplotype or analyze rare mutations.

what is gridded rolling circle nanoballs PCR?

• does not need an amplified template

• used for high-throughput genomic sequencing

• the gDNA is fragmented because it is too big and adequate-sized fragments are selected and linkers are put on them

  • all the fragments have linkers on the ends

• the linkers turn into a circle which is sequenced

• generates ssDNA

what is bridge PCR?

• does not need an amplified template and is isothermal meaning that it stays at one constant temperature to do everything

• not cheap

• not as effective as amplifying unlike PCR

• in 35 cycles, 1000 copies are made but sensitive enough for sequencing

• template annealed to one primer, then denatured with chemicals, chemicals are moved and extended from reverse primers

what is the process of bridge PCR?

• DNA is sheared to manageable fragments and adapters are put onto the sheered DNA

• DNA strands are attached to a cell surface at one end

  • ssDNA will bind to the flow cell adapters in a random pattern

• as the ssDNA flows, the DNA strands will fold over and form a bridge by binding to the complementary primers/adapters

• DNA polymerase binds to the adapter and creates a double strand which is the reverse strand

• the strands are denatured from each other (reverse and forward strand)

  • one adapter on each strand will denature from the flow cell

• the process repeats forming clusters of identical strands

• reverse strands are denatured and washed away

what are the things that ligase chain and branched DNA reaction have in common?

• isothermal

• probe is amplified not the target

• probes will bind adjacent to one another on template

• bound probes are ligated and become templates for the binding of more probes

• better at quantifying amount of target present in clinical samples

  • C. trachomatis, N. gonorrhea, sickle cell mutation

what does it mean if a technique is isothermal? is this an advantage? (written response)

if a technique is isothermal, it means that the technique only needs an environment where it can hold a certain temperature it means that the technique only needs one constant temperature to do everything. it is an advantage as isothermal techniques are faster. Isothermal techniques are also cost-effective because a thermal cycler is not needed.

what is ligase chain reaction?

• probe is amplified and four primers are designed

• when the template is denatured, top strand will have two primers complementary to it that will ONLY amplify it and bottom strand is bound by the two primers that have REPORTERS

  • need to be stuck to each other to excite each other

• after one round of PCR, ligase is added to form the band between two fragments to get stuck together

  • they excite one another to fluoresce

• can be done again to make more target for the next step

what are some applications for LCR?

• can ID gonorrhea and chlamydia very easily from first catch urine

• very sensitive way to detect SNPs

• can detect DNA mutations like sickle cell mutation

  • sickle cell is caused by one nucleotide mutation so a primer is designed around the one nucleotide mutation

  • make the very end of the probe the DNA mutation site and if it is normal or a wild type, it will anneal but if it is the sickle cell mutation it would not anneal

    • the ligase cannot function if the sequence is not a perfect match

what is branched DNA (bDNA)?

• allows signal amplification when working with low sample concentration

  • commonly used to detect viral loads (HBV, HCV, HIV-1)

• need to know the sequence to be able to design a probe to it for amplification of the probe

• works with DNA or RNA

  • targets DNA or RNA released from cells attached to bound capture probe only when denatured

what is the process of branched DNA?

• capture probe is bound to a base that is bought

  • a capture probe extender is designed to be complementary to the capture probe and specific to target

  • the amplifier will be specific to target and the other half will be universal

    • universal portion will recognize a universal amplifier

  • the prongs on the amplifiers will grab onto the signal and will amplify the signal

what are the steps of branched DNA to detect viral load?

• lyse the virus to release its genome

• hybridize the extenders and capture probes

  • one half specific to the target and the other half is universal

• add amplifier and it will start to create antenna

• add different molecule of an unknown sequence that further creates antenna

• add label/reporter that will fluoresce and the degree of fluorescence is amplified

what is the relationship between PCR and template amplification? why amplify?

• PCR amplifies the sequence from the template and the template makes copies of the target sequence

• should amplify to make more of the sample

what is the relationship between PCR and probe amplification? why use probes?

• both detect specific sequences and probes are used to measure the amount of target

• to detect specific sequence of DNA that needs to be analyzed and to ensure it is the target

what is the relationship between PCR and signal amplification? why amplify signal?

• PCR utilizes signal to see how much sample they have and signal amplification can detect low samples

• to detect samples with low-volume

what is nuclei acid sequence based amp (NASBA)?

• isothermal method that does not use Taq and amplifies RNA

• used in clinical, environmental, and food testing applications and HCV and HIV viral loads

• specific for ssRNA

• better than transcription-mediated amplification

• required two target-specific primers

  • real-time fluorescence detection of RNA amplicon generated from NASBA can be accomplished by the incorporation of molecular beacons

• quantification occurs by completing time to positive (TTP) values (analogous to Ct in real-time PCR) compared to a known standard curve

• amplification > 1012 fold in 2 hours

what are the steps for NASBA?

• ssRNA uses primer p2 to turn it into a cDNA double stranded fragment via reverse transcriptase

• add RNase H to degrade the RNA in the RNA:DNA duplex

• use T7 RNA polymerase to turn DNA back into RNA

what is transcription-mediated amplification (TMA)? what are the steps for TMA? what does it work for?

• an isothermal process that allows for direct direction of RNA viruses

• start with an organism that has ssRNA genome and a primer is designed where half of it is universal and the other half is specific to the target RNA genome

  • universal part is used to allow you to use reverse transcriptase to turn it into cDNA

• add RNase H to chew up the RNA, leaving the cDNA

• DNA polymerase creates a second strand of DNA and RNA polymerase converts the universal primer to RNA

• works for tuberculosis, trachomatis, HIV, and CMV

what is the 2nd gen of TMA?

• only uses avian myoblastosis virus (AMV) reverse transcriptase and T7 RNA polymerase

  • has DNase activity meaning it degrades DNA

• isothermal and detects RNA viruses directly and is more sensitive (many RNAs correspond to one DNA)

• primers must have Pol binding site

what is multiple displacement amplification (MDA)?

• not PCR based and uses Φ29 (phi 29) which is a better enzyme than Taq

  • makes a mistake every 10^7 bp so it has a high fidelity but is expensive

• good for whole genome sequencing because it has a high fidelity

• a minute amount of DNA amplifier for genomic analysis and uses random hexamer primers

• Φ29 can do long reading before it stops or falls off (30 C)

  • can extend up to 70 kbp

what are the steps for multiple displacement amplification (MDA)?

• MDA uses random hexamers to bind to all of the gDNA for amplification

  • hexamer creates one fragment and a different hexamer displaces one strand to make a really long read and the other can displace the strand in front

    • each of then are extending the exact same template at the same exact time by picking each other off

• MDA can generate 1-2 micrograms of DNA from single cell with genome coverage of up to 99%

  • products have lower error rates and larger sizes with Φ29

what is loop-mediated isothermal amplification (LAMP)?

• uses multiple primers to the same target and the primers create them

  • uses 4-6 primers recognizing 6-8 distinct regions of targets

• 2 of the primers form loop structures to facilitate subsequent rounds of amplification

  • the loop is good for stability and great to prime off of

• fast —> gets results in 60-75 minutes

• the primers quickly tell you if the target is present or not using the primers that create a loop

• reliable —> strand-displacing DNA polymerase initiates synthesis

what are the uses for LAMP? what are the indicators for positive LAMP?

ID of food pathogens

• dozens of bacterial and fungal food pathogens

positive indicators = white precipitate, liquid is a darker color, glows in UV light

discuss the advantages and limitations of PCR (written response)

One advantage of PCR is that it can detect low viral loads which can diagnose individuals that may have HIV and HCV. Another advantage of PCR is that it is versatile. It can detect viruses and pathogen on different samples like tissues. A limitation of PCR is that it is easily contaminated if not careful which can lead to false negatives or positives.

what has the FDA approved? (written response question)

The FDA has approved tests for human genetics and microbe ID. These tests are nucleic acid bases where the structure, sequence, or expression of DNA or RNA is analyzed. These tests can detect various cancers like leukemia, breast cancer, and can detect chromosomal abnormalities.