MIDTERM FLASHCARDS (combined)

260 nm

Nucleic acid absorb strongly at ____

Polymerase Chain Reaction

What does PCR stands for?

260 and 280

the ratio that gives an estimate of the purity of nucleic acid

260/280 ratio of 1.8-2.0

Pure preparations of DNA

1.8-2.0

ratio of pure preparation of DNA

260/280 ratio 1.8-2.0

260/230 ratio 2.0-2.2

DNA Template

260/280 ratio 2.0

RNA Template

Mg2+ concentration

• 1mM to 5 mM

• Excess - non-specific primer binding

• Two little - low yield

• ^[Mg] high sensitivity, low specificity

0.1-0.6 uM

Primer concentration

5 C - 10 C below melting temp of primers

Annealing temperature

50-500 uM

dnTP concentration

0.5-3.5 units / 50 uL

DNA polymerase

Gradient PCR

• Generate a temperature gradient for the annealing step

• focused on the annealing temperature

Heat Stable DNA Polymerase

• Isolated from hot spring dwelling bacterium

• Not destroyed at 94 C

• Optimal temperature is 72 C

• Error rate 3.0 × 10-4

• Widely used for endpoint PCR

72 C

Optimal temperature for heat table DNA polymerase

3.0 × 10-4

Error rate for Heated Stable DNA Polymerase

Heated Stable Polymerase

Widely used for endpoint PCR

Thermus aquaticus (Taq)

Stabilizes the entire DNA polymerase process

Does not proof read

Error rate 1 per 3,300 bases

Newer polymerase have high fidelity

Problems with Taq

High Fidelity DNA Polymerase

• Hyperthermophilic archaeon

• 3’-to-5’ exonuclease activity

• Excellent for GC-rich templates

• Error rate 1 per 1,000,000 bases

• Applications: cloning, DNA sequencing

Pyrococcus furiosus

What does Pfu stands for?

230

At what absorbance indicates the presence of phenol

280

At what absorbance indicates the presence of protein

Gradient PCR

Optimize the reaction in one PCR run

94 C

Heat Stable DNA Polymerase is not destroyed at what temperature?

3’-5’

Exonuclease activity of High Fidelity DNA Polymerase

High Fidelity DNA Polymerase

Excellent for GC-rich templates

1 per 1,000,000 bases

error rate of High Fidelity DNA Polymerase

Cloning

DNA Sequencing

Applications of High Fidelity DNA Polymerase

Polymerase Chain Reaction (PCR)

In-vitro enzymatic amplification of a specific segment of DNA

Dr. Kary Mullis (1985)

Discovered the Theoretical basis

High temperature

Double-stranded DNA denatures at what temperature?

dsDNA

Exponential increase of _____ produced with increasing cycles

Buffer

Deoxyribonucleoside triphosphates

Magnesium

Polymerase

Primers

Salts

Template DNA

Components for PCR

Buffer

Neutralize DNA polymerase inhibitors present in blood

Deoxyribonucleoside triphosphate (dNTP)

building blocks (monomeric units) of DNA

Magnesium

cofactor of enzyme activity

Polymerase

responsible for elongation of new DNA strands

Primers

DNA fragments that recognize and bind to target DNA segment

Salts

provide the correct ionic strength

Template DNA

double-stranded helix containing genetic code

Denaturation (95 C)

double-stranded DNA (dsDNA) separates into two single strands

Annealing (50-60 C)

• Primer bind to the target DNA sequence

• Red short lines addition of primers to the single strands from DNA after denaturation

Elongation (72 C)

DNA Polymerase extends the primers, synthesizing new DNA strands

Nobel Prize (1993)

What did Dr. Kary Mullis won and what year was it?

increase of dsDNA

Exponential _________ produced with increasing cycles

Rapid detection

Minimal sample required

Useful for strain differentiation

Some pathogens are difficult to cultivate

High analytical sensitivity and specificity

Provides more definitive diagnostic information

Advantages of Amplification Techniques

1000 uL

1mL is equivalent to _____ uL

500 uL

0.5 mL is equivalent to ____ uL

Pathogen

Mutation

Genetic marker

PCR confirms the presence of specific:

Traditional / Endpoint PCR

• uses agarose (from seaweed) gel for detection of PCR amplification at the final phase or endpoint of the PCR reaction

• size-based discrimination only

• Post-PCR processing

Sized-based discrimination

tells presence and size of amplified DNA

Post-PCR processing

• analyze amplified DNA after reaction is complete

• different from qPCR which monitors DNA amplification as it happens

Marker / Ladder

What are these called?

note: numbers inside the red rectangle

Poor precision

Low sensitivity

Low resolution

Problems with Endpoint Detection

<2 logs

dynamic range of Endpoint detection

PCR-RFLP (Restriction Fragment Length Polymorphism)

A molecular technique that combines PCR and restriction enzyme digestion to analyze genetic variations (polymorphisms) in DNA sequences

PCR amplification > Restriction enzyme > Gel electrophoresis

To detect genetic variations using these:

Genetics

Disease detection

Forensic Science

RCP-RFLP is widely used in

Nested PCR

• A modification of traditional PCR

• Improves specificity and reduces non-specific amplification by using 2 rounds of PCR with 2 sets of primers

2 rounds

Round/s needed using Nested PCR

2 primers

How many primers needed for the round/s of PCR in Nested PCR

1st round

• Larger region of DNA is amplified

• Produces a bigger PCR product, but it may contain non-specific DNA (unwanted fragments)

• Result = primary amplicon

Primary amplicon

Result of the 1st round when Nested PCR is used

2nd round

• small portion of the first PCR product is used as a template for the second round

• inner primers are used (designed within the first PCR product)

• specifically amplifies only the correct DNA sequence, filtering out non-specific bands

Multiplex PCR

• Allows multiple DNA targets to be amplified simultaneously in a single reaction

• several primers are used to amplify multiple sequences at the same time

• Efficient and cost-effective, enabling the detection of various genes or pathogens concurrently

Multiplex Real-time PCR

• Advanced version of Multiplex PCR

• multiple DNA targets are amplified and detect in real-time using fluorescent probes

Fluorescent probes

Multiple DNA targets are amplified and detected in real-time using _________

Lag Phase - Exponential Phase - Linear Phase - Plateau Phase

4 stages of qPCR Amplification curve

Real-time PCR (qPCR)

• Variation of endpoint PCR

• Amount of amplification product is measured at each reaction cycle via fluorescent dyes, probes

• Required gel electrophoresis to analyze results after the reaction

• It is faster, more sensitive and widely used in disease diagnosis, genetic research and forensic science

Fluorescent dyes

Probes

Amount of amplification product is measured at each reaction cycle via:

Threshold cycle (Ct)

the product can be detected above the background

Ct (Cycle Threshold) value

The cycle number where fluorescence crosses the detection threshold

Lower Ct value - Higher DNA Concentration

Understanding qPCR Results:

more starting DNA

Higher Ct value - Lower DNA Concentration

Understanding qPCR Results:

less starting DNA

No Ct value - No target DNA

Understanding qPCR Results:

present or below detection limit

Relative Quantification

• Expression of a gene in one sample is compared to the expression of the same gene in another sample

• Results = fold change

Absolute Quantification

Sample of known quantity are serially diluted and amplified to generate a standard curve

Probe-based

• Uses double-labeled sequence specific probes

• Composed of an oligonucleotide with fluorescent dye and quencher

• Fluoresces only when the probe hybridizes to specific target

Fluorescence Resonance Energy Transfer

what does acronym FRET stands for?

Intercalating Dye

• non-specific DNA binding dye

• fluoresces upon intercalation into dsDNA

RVR

Viral load not detected at 4 weeks of treatment

EVR

>2 log decrease from baseline viral load or not detected at 12 weeks treatment

SVR

Viral load not detected 6 months after end of treatment

Melting Temperature

function of product length and base composition

Melting Curve

Charts the change in fluorescence observed when dsDNA with the dye dissociates or melts into ssDNA as the temp is raised

Melting Curve Analysis

• If the target is 1 defined genetic sequence, it should have 1 specific Tm

• Target peak should be narrow, symmetrical, devoid humps or splits

Reverse Transcription PCR (RT-PCR)

Amplify an RNA target sequence

HIV

Dengue

SARS-COV

RT-PCR is used to detect:

Double-stranded nucleic acid

In RT-PCR, RNA sequence is converted to a _________ sequence using reverse transcriptase

One-Step

Two-Step

Protocol used of RT-PCR

One-step

• Reaction takes place in a single tube

• use of sequence-specific primers

Two-step

• random hexamers

• oligo dT

• gene-specific primers

10.0 uL

Total reaction volume for cDNA Synthesis

RNA Extraction - Reverse Transcription - cDNA Synthesis - PCR Amplification - Agarose Gel Electrophoresis - Image Analysis

Steps in RT-PCR:

RNA Extraction

Isolating RNA from a sample

Reserve Transcription

RNA -> complementary DNA (cDNA)

cDNA Synthesis

Producing the DNA Template for PCR

PCR Amplification

Running PCR to amplify specific DNA sequence

Agarose Gel Electrophoresis

Running the amplified DNA on a gel to separate by size