Oral Boards 4- Adfs

Front 1

Quant instrument maintenance schedule

Back 1

-Monthly:

1. Background Calibration

2. ROI Inspector

3. Check PC & Defragment

-Quarterly:

4. Hardware Test

5. Lamp

-Semi-annualy:

6. ROI Calibration

7. Pure Spectral Assay

Front 2

What are the phases of amp process in Quant?

Back 2

1. Exponential/Geometric amp

2. Linear amp

3. Plateau region

Front 3

What does IPC stand for?

Back 3

internal PCR control

Front 4

Describe the plateau phase of quant

Back 4

-signifies depletion of critical reagents and is know as the plateau

-amp process ceases

Front 5

Describe the exponential phase of quant

Back 5

-theoretical doubling of amplicons with each cycle

-baseline determined by measuring background fluorescence signal (noise)

-baseline establishes threshold

Front 6

Explain cycle threshold

Back 6

-the point in which the level of fluorescence exceeds the threshold

-Ct is lower for a sample with higher initial concentration (higher Ct for a sample with lower concentration DNA)

Front 7

What is the size of the human telemorase reverse transcriptase target gene?

Back 7

62 bases

Front 8

How is baseline determined? What does it establish?

Back 8

-baseline determined by measuring background fluorescence signal (noise)

- baseline establishes threshold

Front 9

What is the purpose of DNA quantitation?

Back 9

-To estimate the amount of HUMAN DNA present in the sample

-helps determine the target amount of DNA needed to get a good profile

-PCR assays work best in a fairly narrow range of DNA

-target is 1.0 ng of DNA

-too little= may fail to amp/dropout

-too much= off scale peaks/blowout

Front 10

Back 10

A. Exponential product growth

B. Linear product growth

C. Plateau phase

D. Cycle number

E. Threshold

F. Normalized fluorescence

Front 11

Describe the linear phase of quant

Back 11

-the consumption of 1 or more PCR reagents during amp will impede the efficiency causing the onset of linear phase

-product amp slows down, yielding an inconsistent ratio of input DNA to product

-not commonly used in data analysis

Front 12

Purpose of minor groove binder?

Back 12

-linked to 3' end of probe

-forms extremely stable duplexes with ssDNA targets, allowing shorter probes to be used

-increase melting temperature without increasing probe length

-increase sensitivity because it's more specific for single base mismatches and fluorescence quenching more efficiently

Front 13

What is Rn? What is ΔRn?

Back 13

Rn: normalized reporter is the the ratio of the fluorescence emission intensity of the reporter dye to the fluorescence emission intensity of the passive reference dye

Δ Rn: normalization of Rn obtained by subtracting the baseline: Δ Rn= Rn - baseline

Front 14

What is the y-intercept in quant data?

Back 14

-theoretical value for nucleic acid concentration

-indicates the expected Ct value for a sample with quantity= 1 ng/uL

Front 15

A. How many pg of DNA are in 1 haploid cell? 1 diploid cell?

B. How many cells are in 1 ng of DNA in a haploid/diploid cell?

C. How many copies in a diploid single-copy locus with concentration of 23 pg? haploid single copy locus?

Back 15

A. 3 pg (haploid), 6 pg (diploid)

B. 333 cells/333 copies (haploid)

167 cells/333 copies (diploid)

C. 3-4 copies (haploid), 7-8 copies (diploid)

Front 16

What is the amplification plot for quant?

Back 16

-plot of the fluorescence signal v. cycle number

-reactions are characterized by the point in time during cycling when amplification of PCR product is first detected

-the higher the starting copy number of the nucleic acid target, the sooner a significant increase fluorescence is observed

Front 17

A. true negative

B. invalid result

C. disregard IPC result

D. partial PCR inhibition

Back 17

A. quant: no amp & IPC: amp

B. quant: no amp & IPC: no amp

C. quant: amp (low Ct, high ΔRn) & IPC: no amp

D. quant: amp (high Ct, low ΔRn) & IPC: no amp

Front 18

Purpose of the IPC

Back 18

-troubleshoot

-detects inhibitors, contamination, true negative samples, or failure of run

-should give amp product even if no sample DNA is present

Front 19

5' Nuclease Assay Process: How does probe work? How is fluorescence detected?

Back 19

-FRET (fluorescence resonance energy transfer)

-multicopy sequence specific probe (TaqMan): binds to targeted template

-probe has 2 dyes: reporter (5' end) and quencher (3' end)

-close proximity of 2 dyes allows energy transfer to occur between the two; quencher suppresses fluorescence of reporter

-probe hybridizes to complementary target: is then cleaved by 5' exonuclease activity of DNA polymerase

-cleavage separates reporter dye from NFQ: increases reporter dye fluorescence at each PCR cycle: amount of fluorescence measured at each cycle

Front 20

It is recommended that samples exhibiting a RT-PCR quant greater than --A-- should be diluted and requanted as the RT-PCR data may have exceeded the --B-- of the standard curve.

Back 20

A. 100 ng/uL

B. linear range

Front 21

Describe denaturation, annealing, and extension for quant reaction. How many cycles?

Back 21

1. denaturation: 94-95 C, dsDNA becomes ssDNA

2. annealing: temp is decreased, primers and probes anneal to template DNA, no fluorescence detected, NFQ is absorbing reporter dye energy according to FRET

3. extension: temp is increased, Taq extends forward primer by adding complementary dNTPs, Taq cleaves probe, reporter dye fluoresces

Front 22

Full name of quant probe

Back 22

Taqman Minor Groove Binding Probe

Front 23

Components of IPC assay

Back 23

-IPC template DNA (synthetic sequence, not found in nature)

- One Taqman MGB probe labeled with VIC dye for detecting amped IPC DNA: linked to 5' end of probe

-NFQ on 3' end of probe

-MGB on 3' end of probe

Front 24

What is Ct? What is the equation for Ct?

Back 24

-cycle threshold: intercept at which the amp signal exceeds the background signal and crosses the threshold

Ct= m [log(Qty)] + b

(where m is the slope and b is the y-intercept)

Front 25

What is the correlation coefficient?

Back 25

-R^2

-relationship between the standard concentrations and concentrations calculated based on the standard curve

-closeness of fit between the standard curve regression line and individual Ct data points of the standards

* should NOT be lower than 0.98

Front 26

Is the probe used for quant single copy or multicopy sequence?

Back 26

multicopy sequence specific

Front 27

What are the 3 human specific target loci in quant trio?

Back 27

1. Small Autosomal: primary quant target: 75 to 80 bases in length: helps determine sample concentration

2. Large Autosomal: greater than 200 bases in length: aids in determining sample degradation

3. Y-chromosome: 75 to 80 bases in length: helps determine male sample concentration

Front 28

Does quant trio use multi-copy or single-copy target loci?

Back 28

-multi-copy

-each target loci consist of multicopies dispersed on various chromosomes

Front 29

What are the four QT 5' nuclease assays?

Back 29

-two separate target-specific human assays

1. one with a short PCR amplicon

2. one with a long PCR amplicon

-target specific human male DNA assay

-internal PCR control (IPC) assay

Front 30

Explain small autosomal human target in QT

Back 30

-amplicon length= 80 bases

-ploidy= diploid

-copy number= multicopy

-dye= VIC dye

-quencher= MGB quencher

Front 31

Explain large autosomal human target in QT

Back 31

-amplicon length= 214 bases

-ploidy= diploid

-copy number= multicopy

-dye= ABY dye

-quencher= QSY quencher

Front 32

Explain the male human target in QT

Back 32

-amplicon length=75 bases

-ploidy= haploid

-copy number= multicopy

-dye= FAM dye

-quencher= MGB quencher

Front 33

Explain the internal PCR control in QT

Back 33

-amplicon length= 130 bases

-ploidy= n/a

-copy number= synthetic IPC template is included in the primer mix

-dye= JUN dye

-quencher= QSY quencher

Front 34

With QT how do you indicate DNA degradation?

Back 34

-by comparing the ratio of the large autosomal target with the ratio of small autosomal target quantification

Front 35

What are the IPC components in QT?

Back 35

1. IPC template DNA (sequence no found in nature)

2. Primers for amplifying the 130 base IPC template DNA

3. TaqMan probe dye-quencher (JUN dye with QSY quencher)

Front 36

Explain 5' Nuclease Assay process in QT.

Back 36

-takes place in every cycle of PCR amplification and doesn't interfer with exponential accumulation of product

-4 steps:

1. polymerization

2. strand displacement

3. cleavage

4. completion of polymerization

Front 37

What happens to the 5' Nuclease Assay in polymerization?

Back 37

-Taqman probe anneals specifically to a complementary sequence between the forward and reverse primer sites

-because the reporter dye and quencher are close in proximity, there is suppression of reporter fluorescence (due to energy transfer)

Front 38

What happens to the 5' Nuclease Assay in strand displacement?

Back 38

-TaqMan probe is displaced by DNA polymerase adding complementary sequence

Front 39

What happens to the 5' Nuclease Assay in cleavage?

Back 39

-Taq DNA polymerase enzyme cleaves probes that are hybridized to the target

-cleavage separates reporter dye from quencher

-results in increase in fluorescence by reporter

-increase in fluorescence signal only occurs if the target sequence is complementary to probe and is amplified (so non-specific amplification isn't detected)

Front 40

What happens to the 5' Nuclease Assay in the completion of polymerization?

Back 40

-polymerization of strand continues, but because the 3' end of the probe is blocked, there is no extension of the probe during PCR

Front 41

Explain steps which occur on the 7500 RT-PCR system for fluorescence detection

Back 41

1. a tungsten-halogen lamp directs light to each well on the reaction plate: the light excites the fluorescent dyes in each well on the plate

2. the CCD camera detects the fluorescence emission

3. the 7500 system obtains the fluorescence emission data from the CCD camera, and uses the data obtained form the pure-dye calibration to distinguish the individual contribution of each dye in the collective fluorescence as gathered by the instrument during a run, and applies data analysis algorithms

Front 42

How to make the 5 standards in QT?

Back 42

STD 1: 50.000 ng/uL: 10 uL (of 100 ng/uL stock) + 10 uL TE: dilution factor of 2X

STD 2: 5.000 ng/uL: 10 uL STD 1 +90 uL TE: dilution factor of 10X

STD 3: 0.500 ng/uL: 10 uL STD 2 + 90 uL TE: dilution factor of 10X

STD 4: 0.050 ng/uL: 10 uL STD 3 + 90 uL TE: dilution factor of 10X

STD 5: 0.005 ng/uL: 10 uL STD 4 + 90 uL TE: dilution factor of 10X

**made with QuantTrio THP DNA Standard

Front 43

The STD curve for the QT kit is linear between what concentrations?

Back 43

5 pg/uL to 100 ng/uL

*be cautious with anything less or greater

Front 44

Explain the R^2 value

Back 44

-measure of the closeness of fit between the STD curve regression line and the individual Ct data points of quant STD reactions

-value of 1.00 is a perfect fit

-R^2 value greater than or equal to 0.99 indicates a close fit between the regression line and Ct points

Front 45

What does the slope indicate in the regression line in quant?

Back 45

-indicates the PCR amplification efficiency for the assay

-slope of -3.3 indicates 100% amplification efficiency

Front 46

The appropriate slope ranges for each target in QT?

Back 46

-small autosomal: -3.0 to -3.6

-large autosomal: -3.1 to -3.7

-Y target: -3.0 to -3.6

- if slopes fall outside range, interpret with caution

Front 47

What does the y-intercept indicate in quant regression line?

Back 47

-indicates the EXPECTED Ct value for a sample with a quantity of 1 ng/uL

Front 48

The IPC helps distinguish between a true negative and reaction affected by what?

Back 48

-presence of PCR inhibitors

-assay setup

-a chemistry or instrument failure

Front 49

For positive amp, what Ct value will it be?

Back 49

-Ct value for the target will be below 40

Front 50

What can increase the IPC Ct value?

Back 50

-presence of PCR inhibitors

-and/or concentrations of DNA

Front 51

What could cause complete amp failure?

Back 51

-incorrect thermal cycling

-incorrect formulation of PCR reaction mix

-severe PCR inhibition

Front 52

What indicates suppressed amplification?

Back 52

High Ct value and Low ΔRn value

Front 53

How sensitive is Quant Trio?

Back 53

-can reproducibly quantify 5 pg/uL of human genomic DNA

-when 2 uL of a sample is loaded with 5 pg/uL of DNA, the well contains about 1.5 diploid human genome

Front 54

When do we start seeing stochastic effects?

Back 54

concentrations below 5 pg/uL

Front 55

What options do you have if quant indicates there is insufficient DNA?

Back 55

1. re-extract the sample and re-quant

2. concentrate the sample and re-quant

Front 56

How is the degradation index calculated?

Back 56

(small autosomal DNA concentration)/(Large autosomal DNA concentration)

Front 57

What affects the degradation index?

Back 57

-degree of degradation of the large autosomal target DNA

-presence of PCR inhibitors (especially PCR inhibitors that negatively affect the LA target more so than the SA target)