Chapter 7: Nucleic Acid Amplification

Internal control

(amplification control) a second set of primers and an unrelated target are added to the reaction mix to demonstrate that the reaction is working even if the test sample is not amplified.

Noncomplementary extensions or tails can be added to the 5′ end of the primer sequences to introduce useful additions to the final PCR product, such as: (3)

**Termed Tailed primers: can happen on bth ends 1. restriction enzyme sites 2. promoters 3. binding sites for other primers

Tth polymerase was isolated from? Describe

- Thermus thermophilus 1. has reverse transcriptase activity (RNA starting template0

Taq polymerase was isolated from? Describe the enzyme (2)

-thermophilic bacterium (thermus aquaticus) 1. added at beginning of methid 2. maintains activity throughout heating and cooling

Describe oligo dT permers?

1. 18-bases long 2. single stranded 3. polyT sequences (prime cDNA from mRNA with ployA tails)

Give (4) Key Points on Deoxyribonucleotide Bases

1. 4 deoxyribonucleotide triphosphate (dNTPs) - adenine, thymine, guanine, cytosine 2. building blocks of DNA 3. dNTPs are added to the synthesis reaction in concentrations that will accommodate the exponential increase in the copies of template. 4. Standard procedures require 0.1 to 0.5mM concentration of each nucleotide.

What is a primer dimer?

1. Are PCR products that are double the size of primers. 2. They result from the binding of primers onto each other through short (2-3 base) homologies at their 3′ ends and the copying of each primer sequence

What are the accessory PCR Buffers used for Optimization? (4)

1. Bovine serum albumin 2. Dithiothreitol 3. Formamide 4. Chaotropic agents

What are subjected to amplification program? (5)

1. DNR template 2. primers 3. nucleotides 4. polymerase 5. buffers

Application of RT-PCR

1. Expression profiles (rRNA, mRNA) 2. Detect genes with long introns 3. Detect microorgansims with RNA genomes

Annealing temperature? (2)

1. If the temperature is too high, binding of the primer to the target will not be stable enough for amplification to occur. 2. If the temperature is too low, then more partial primer-target mismatches will occur causing more non-specific products

What are different modifications of PCR/Target Amplification? (5)

1. Multiplex PCR 2. Reverse Transcriptase PCR (RT-PCR) 3. Real-Time PCR (qPCR) 4. Nested PCR 5. Arbitrarily Primed PCR

What are the 3 different types of Nested PCR?

1. Nested 2. Semi-Nested 3. Tailed first round primers

Describe Primer length (2)

1. Optimal length for a PCR primer is 18-22 bp. 2. Long enough for adequate specificity and short enough to bind easily to the template at the annealing temperature.

PCR Primer Design (7)

1. Primer length 2. Primer melting temperature 3. Forward and reverse primers 4. GC content 5. Primer secondary structures 6. Amplicon size 7. Primer specificity

What affect the Tm of primers? (5)

1. Reaction conditions 2. salt concentrations 3. mismatches 4. template condition 5. secondary structure

What are components of one step PCR? (3)

1. Tth DNA Polymerase (RT activity) 2. property mixtures 3. sequestered (hot-start) DNA Polymerase ~ RNA added directly to PCR ~ 45-50C for 30 to 40 min

Give a brief description of primers

1. contain sequences homologous to site flanking region 2. ssDNA 3. 20-30 bp long 4. forward & reverse primers are started at 5'

What is the three step process of PCR?

1. denaturation ( 90-96C; 20-60 sec) 2. annealing (50-70C; 20-90 sec) 3. Extension (70-75C; 10-60 sec)

Challenges of Multiplex PCR?

1. different sequences may not amplify with the same efficiency 2. primers need to be distinct to not interfere with each other 3. different sized products may be difficult to separate in analysis

Best Template (3)?

1. free of contaminating proteins 2. without nicks or breaks 3. less GC content = less secondary structures

what are psoralens?

1. intercalates between bases in the presence of long-wave UV light (T,U, C) in DNA chain. 2. prevent denaturation and amplification of the treated DNA

Like PCR with DNA Polymerase, RT-PCR need to be primed. What the primers? (2)

1. oligo dT primers 2. random hexamers

What are challenges of one-step PCR? (3)

1. poor specificity 2. low yield 3. system stability

Application of Multiplex PCR? (4)

1. respiratory viruses 2. sexual transmitted organisms 3. accurate detection of a single target 4. carrier of antimicrobial resistant gene

How to prevent primer dimers?

1. use less primer 2. play with MgC12 3. use more template DNA 4. adjust annealing temperature 5. redesign primers

Magnesium concentrations used is?

1.5nM

For routine clinical analysis, _____________ of DNA is usually used.

100 ng to 1 ug. Lesser amounts are required for more defined template preparation such as cloned target

Describe the second step

2 oligonucleotides will prime the synthesizesis of DNA anneal to complementary sequence on the template. (hybridize). Primers dictate what will be amplified.

Good PCR primers generally have a G/C content between ?

40 and 60%.

10 mM Tris-HCL maintains proper pH at?

8-9.5 pH

The optimal pH for PCR is 8.4, however for long templates a higher pH of ________ may be suggested.

9.0

What does AFLP stand for?

Amplified Fragment Length Polymorphism; analysis of polymorphic DNA by amplification of selected regions and resolving the amplified products by size

Describe Rapid PCR?

Are designed to work with very small sample volumes in chambers that can be heated and cooled quickly by changing the air temperature surrounding the samples.

What is the calculation for PCR Master Mix Creation?

Ci)(Vi) = (Cf)(Vf) Ci= initial concentration Cf= final concentration Vi= initial volume Vf= final volume

Detection of RNA Virus such as _________, is responsible for severe acute respiratory syndrome (SARS).

Coronavirus

When does DNA synthesis occur?

During the extension step

The first successful amplification was a short fragment of?

Escherichia coli plasmid, pBR322

Explain the last step of PCR

Extension; DNA polymerase catalyzes the formation of the phosphodiester bonds between incoming dNTP determined by hydrogen bonding to the template (A/T/G/C) and the base at the 3' end of the primer.

Forward primer hybridizes ______ to the sequence being amplified. Reverse primer hybridizes _______ to the sequence being amplified.

Forward primer hybridizes 5' Reverse primer hybridizes 3'

Fragile X Syndrome at 5' to the FMR-1 gene is an example of?

GC-rich target

_________ magnesium concentrations gives a higher yield, but also a __________

Higher; lower specific.

Define Multiplex PCR

It is a variation of PCR that enables simultaneous amplification of many targets of interest in one reaction by using more than one primer.

The four dNTP concentrations should be higher than the estimated ?

Km of each dNTP (10-15 mM, the concentration of substrate at half maximal enzyme velocity).

Fromamide

Lowers the denaturation temperature of DNA with high secondary structure

what are amplicons?

Millions of identical copies of the original or target DNA made in 2 hours

_______ is especially useful in typing or identification analyses.

Multiplex PCR

Negative Control

No DNA, ensures that the reaction mix is not contaminated from a previous run a.k.a contamination control or reagent blank

Does tailed first round primers increase sensitivity and specificity like nested?

No, it only increases sensitivty

What measures are taken for contamination control?

Physical: 1. separate pre-PCR & post-PCR areas 2. positive airflow, airlocks 3. gowns/gloves dedicated Chemical: 1. UV light for pre-pcr 2. 10% bleach 3. dUTP-UNG

What is PCR?

Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify a particular DNA sequence across several orders of magnitude, generating thousands to millions of copies.

What are monovalent cations used in Buffers and their function? (2)

Potassium chloride & Ammonium sulfate - Affect denaturing and annealing temperatures of the DNA -An increase in salt concentration makes longer DNA products denature more slowly than shorter DNA products during the amplification process, so shorter molecules will be amplified at a greater degree

Challenges of RT-PCR

RNA is vulnerable to degradtion

What enzyme is recommended for allele-specific PCR and for amplification of regions with High GC content?

Stoffel Fragment, 1. a modified polymerase enzyme that has twice the half life at high temp of taq polym. 2. broader range of optimal MgCl2 concentrations 3. lacks N-terminal 289 amino acid & 3' to 5' exonuclease activity

What is Sequence-Specific PCR?

The basis for human leukocyte antigen allele analysis in tissue typing. (Design foward & reverse primers to end with 3' base complimentary to the mutant sequence)

What is the Tm formula definition?

The temperature at which half of the sequence is double-stranded and half is single stranded. Tm = 4(G + C) + 2 (A + T)

Describe the Reverse-Transcriptase (RT) PCR method

This originates from RNA virus, and is used to first produce a RNA-DNA Hybrid. Then, a hairpin structure on the end of the DNA strand primes itself for DNA polymerase to produce dsDNA or *cDNA*

Describe Nested PCR method

Two pairs of primers used in 2 rounds of PCR. Second pair binds inside the binding sits of the first pair (reduces products due to mis-priming).

_________ catalyzes single- and double-strand breaks in the DNA that will then interfere with replication

UV light

What is the proofreading enzyme that allows Taq or Tth polymerase to generate larger products over 30,000 bases in length?

Vent Polymerase

Too few Mg2+ ions lower enzyme efficiency, resulting in?

a low yield of PCR product

What is a amplification program?

a specified number of cycles that are divided into steps that are held at particular temperatures for designated times. The temperature determines the reaction that will occur.

what does fidelity means?

accurate copying of template

Positive controls ensure that the enzyme is?

active the buffer is optimal, the primers are priming the right sequences, and the thermal cycler is cycling appropriately

uracil-N-glycosylase (UNG)

added to reaction mix, will degrade any nucleic acid containing uracil, such as contaminating PCR product from previous reactions. A short incubation period is added to the beginning of the PCR amplification program, usually at 50C for 2-10 minutes to allow the UNG enzyme to function.

PCR is the first and prototypical method for ?

amplifying target nucleic acid

What is crucial for specificity ?

annealing

Describe tailed-first round primers?

are 5' sequences complementary to a single set of second round primers that are added to all the first round products

Whay is dUTP-UNG system routinely used in real-time PCR?

because the contaminate will not be distinguishable from the desired amplicon by gel electrophoresis.

bovine serum albumine (bsa)

binds inhibitors and stabilizes the enzyme

Is the template single or double stranded?

both can occur

What is the cause of mispriming?

creates an unintended product that could interfere with subsequent interpretation or mutation analysis. It can also occur in regions unrelated to the intended target sequence.

What serves as the template?

existing dsDNA

Lower Mg2+ concentrations are desirable when

fidelity of the PCR is critical

__________ magnesium concentrations increases specificity, but will ____________.

lower; decrease enzyme activity

PCR methods are capable of detecting multiple targets in a single run (_________), using RNA templates (__________), or such amplified products as templates (_________) and quantitating starting template (quantitative PCR, or ___________).

multiplex PCR; reverse transcriptase PCR; nested PCR; real-time PCR

Tris-buffer and accessory buffer components are also important for _______________ and ______________.

optimal enzyme activity and accurate amplification of the intended product.

Contamination is controlled both ___________ and _________.

physically and chemically

What determines specificity of the PCR?

primers

Primers are analogous to the ___________ in blotting and hybridization procedures.

probes

Define amplicon

product of a PCR reaction

Dithiothreitol (DTT)

provides reducing conditions that enhance enzyme activity

Thermal cyclers or thermocyclers were thus designed to rapidly and automatically ___________ through the required incubation temperatures, holding at each one for designated periods.

ramp (change)

The highest cDNA is achieved with?

random hexamers and decamers

Chaotropic agents

reduces secondary structures to allow for polymerase extension through difficult areas

Denaturation

sample heated to 94-96C (sec-minutes) > dsDNA sep to 2 strands

Nested PCR utilizes multiple rounds of PCR to increase?

sensitivity and specificity to the target regions

Describe semi-nested PCR method

similar to nested but during the second round only one primer is unique (inside) and the other remains on the outside.

Describe dUTP-UNG system

substitution of dTTP with dUTP in the PCR reagent master mix, which will result in incorporation of dUTP instead of dTTP into the PCR product.

Describe Real-time PCR

systems are equipped with fluorescent detectors to measure PCR product as the reation proceeds

Define amplification control

template sequences that are always present; used to confirm the functional competence of the reaction mix

The polymerase will not form a phosphodiester bond if ?

the 3′ end of the primer is not hydrogen-bonded to the template

Overly high Mg2+ concentrations promote misincorporation and thus increase _____________________

the yield of nonspecific products

The Internal control or amplification control is critical to distinguish between?

true negatives for the sample and an amplification failure (false-negative)

PCR essentially duplicates the in ________ replication of DNA in _______, using the same components (Table 7.1) to replicate DNA as the cell does in vivo

vivo; vitro

How to recognize a primer dimer?

when you're expecting 1 band on your gel and you visualize 2

Why is cDNA often used for sequences?

you can determine the coding region without long stretches of introns complicating the analysis

Tm difference between the forward primer and reverse primers should be?

≤ 5