Module 9

Question 1

Polymerase Chain Reaction

Answer 1

-amplify specific DNA sequences
-need primers, reactions mix, amplicon

Target (Template) DNA : The DNAextracted from the specimen

dNTPs (deoxyribonucleoside triphosphates-The “building blocks” added to ss of DNA by Taq polymerase to make new ds of DNA

Primers (Forward & Reverse):
2 short sequences of complementary DNA or RNA (20 base pairs)
* Primers determine the specificity of the PCR reaction
* The distance between the primer binding sites will determine of the size of the PCR product

DNA polymerase:
Enzyme in cells that use existing single strands of DNA as a template to make new double strands of DNA
Reads the template and adds nucleotides one at a time
In PCR use Taq polymerase because it does not denature at the high temperatures required in each PCR cycle

• Taq: Thermus aquaticus (most commonly used)
• Sequenase: T. aquaticus YT-1
• Restorase (Taq + repair enzyme

Question 2

PCR STEPS

Answer 2

Denaturation 90–96oC * 20 seconds
-temperature increased to separate the DNA strands

Annealing 50-65 1-2min
Temperature decreased to allow primers to base pair to complementary DNA template

Extension 68-72 10-15 min
Polymerase extends primer to form nascent DNA strand

PCR Product = Amplicon
The number of amplicons = 2N where N = the number of cycles

Question 3

Automation of PCR

Answer 3

The thermal cycler changes temperature in a block or chamber holding the samples
* Thermostable polymerases are used

Question 4

Interpretation of PCR Results

Answer 4

Easiest way to visualize PCR product is by agarose gel electrophoresis
-Nucleic acids or proteins separated based on size, charge and shape by an electric charge (from – to +)
-Visualized by staining with dye (fluorescence)
-Compared to a ladder (size standard ruler) included on one lane of the gel

*No product should be present in the reagent blank
* Misprimes may occur due to nonspecific hybridization of primers
*Primer dimers may occur due to hybridization of primers to each other

Question 5

Avoiding Mis-Primes & Primer Dimers

Answer 5

• Use proper annealing temperature
• Design primers carefully
• Adjust monovalent cation concentration
• Use hot-start method: prepare reaction mixes on ice, place in
  preheated cycler, or use a sequestered enzyme that requires an initial heat activation

Question 6

Primer Design

Answer 6

• Avoid inter/intra strand homologies
• Tm of forward primer = Tm of reverse primer
• G/C content of 20% to 80%; avoid longer than GGGG
• Product size (100 to 700 base pairs)
• Target specificity

Question 7

Product Cleaning

Answer 7

• Purpose: Removes all reaction components as well as misprimes and primer dimers
• Gel elution
• Solid-phase isolation of PCR product (example, spin columns)
• DNA precipitation

Question 8

Contamination Control

Answer 8

• Any molecule of DNA containing the intended target sequence is a
  potential source of contamination
• Most dangerous contaminant is the PCR product from the previous
  reaction

Physical separation (air-locks, positive air flow), UV, 10% bleach

Question 9

PCR Modifications:

nested primer

tailed primer

Answer 9

nested: product of the first amplification reaction is used as the template for the second PCR

tailed: contains at the 5’ side, the sequence of the gene you want to amplify. controls sequence ensuring the cloning into vectors

Question 10

Quantitative PCR (qPCR)

Answer 10

-qPCR combines PCR amplification and detection into a single step.
-PCR amplification products are assayed as they accumulate rather than at the end of the procedure
-Positive result can be observed while assay is still running
-Uses a fluorescent reporter dye or a probe labelled with a fluorescent dye
-The Thermocyler used will also have a UV light source to excite the reporter & a camera to detect increasing fluorescence at each cycle of amplification

• PCR product grows in an exponential fashion (doubling at each cycle) a lag phase, a log phase, a linear phase, and a stationary phase
• The length of the lag phase is inversely proportional to the amount of starting material

Question 11

qPCR – Detection Systems

Answer 11

• DNA-specific dyes
• Ethidium bromide
• SYBR green
• Hybridization probes
• Cleavage-based (TaqMan)
• Displaceable (molecular beacons, FRET)
• Primer-incorporated probes

Question 12

qPCR – SYBR Green

Answer 12

*Fluorescent dye that binds to any double strand of DNA
*less specific than Taqman
* Binds minor groove of double stranded DNA
* Product can be further tested in
a post- amplification melt curve
where sequences have
characteristic melting temperatures

Question 13

qPCR – TaqMAN

Answer 13

• short sequence of DNA specific to a section of the target DNA.
  -fluorescent dye on its 5’ end and quencher on its 3’ - closed no fluorescence
  -hybridizes between the primer sites and get digested via exonuclease activity of DNA polymerase
  -glows after getting digested
  The amount of signal depends on the number of targets amplified

Question 14

qPCR – Fluorescence Resonance Energy Transfer (FRET) Probes

Answer 14

They bind adjacent to one another on the target
* Allowing the donor to transfer energy to the reporter dye
* The amount of signal depends on the amount of target present

Question 15

qPCR – Molecular Beacons

Answer 15

• Molecular beacons bind target to produce signal
• The reporter (R) and quencher (Q) are on opposite ends of the probe and held together by about 5 bp of homology when not bound to the target bound to each other like a key shape and then they become linear when binding DNA
• In the presence of target sequences, the probe binding overcomes the short-end homology, releasing the reporter from the quencher

Question 16

qPCR – Scorpion Probes

Answer 16

• Scorpion primer-binding site is added to any target using a 5’ tailed primer in an initial PCR
  reaction
  -fluorescent signal is covalently attached to the PCR product
• allowing subsequent analysis, for instance, by capillary electrophoresis

Question 17

Point Mutations

Answer 17

• Point mutation involve one or few base pairs
• Do not always have a phenotypic effect

silent mutation - change in DNA sequence does not affect sequence of aa

conservative mutation - aa substitution with another aa having no impact

non conservative - aa substitution has different properties than original ie sickle cell

nonsense - change in DNA that causes a protein to terminate or end translation early

frameshift - insertion or deletion of nucleotide bases that are not multiples of 3 because a cell reads a genes code in multiples of three

Question 18

Biochemical Methods to detect mutations

Immunoassay
HPLC
– Gas Chromatography

Answer 18

Immunoassay
* Antibodies that detect mutant or wild-type proteins
* Immunohistochemistry in tissue

HPLC
*Uses solid and liquid phases to separate particles by size, charge, or chemical characteristics

Gas Chromatography
* Samples are vaporized and
separated in a gas phase
-MALDI-TOF using ddNTPs

Question 19

Nucleic Acid-Based Detection Methods :* Hybridization based

Single-Strand Conformation
Polymorphism (SSCP)

Melt-Curve Analysis

Array Technology

Answer 19

Single-Strand Conformation Polymorphism (SSCP)
- Single-strand conformational polymorphism (SSCP), melt curves, array technology
-one base affects folding
-strict temp requirements

Melt-Curve Analysis Based on sequence effect on Tm
* Dye specific to double-stranded DNA (SYBR green) or FRET probes will fluoresce when bound to DNA
* Denaturation of DNA to single strand will result in loss of fluorescence
* The speed of drop of fluorescence (dF) will be maximal at the Tm
* Every sequence has a characteristic Tm
* Melt-curve Tm indicates which sequence is present

Array Technology
*reverse-dot-blot methods
* used to investigate multiple genomic sites simultaneously
* Unlabeled probes are bound to substrate
* Specimen DNA is labeled and hybridized to immobilized probes

Question 20

Nucleic Acid-Based Detection Methods : Sequencing-based

Sequence-Specific Primer PCR (SSPPCR)

Allelic Discrimination

Answer 20

Sequence-Specific Primer PCR (SSPPCR)
* PCR primer extension requires that the 3’ base of the primer is
complementary to the template
* Used to detect point mutation
* Presence or absence of product interpreted as presence or absence of the mutation

Allelic Discrimination
* Uses fluorescently labeled probes
* Quantitative PCR (qPCR) technology
* Generates “color” signal for mutant or normal sequence
* Performed on real-time PCR instruments

Question 21

Nucleic Acid-Based Detection Methods : Cleavage-based

Restriction Fragment Length
Polymorphism (RFLP)

Answer 21

Restriction Fragment Length
Polymorphism (RFLP)
* Restriction enzyme site recognition detects presence of sequence changes
* For example, G→A change creates EcoR1 site

Question 22

Heteroduplex Analysis with Single-Strand-Specific
Nucleases

Answer 22

• Uses nucleases that cut singles tranded bubbles in heteroduplexes
• Region of interest is amplified by PCR or transcribed
• The double-stranded (DS) product is denatured and renatured with or without added normal product
• Renatured duplexes are digested with nuclease
• Products are observed by gel
  electrophoresis

Question 23

DNA Sequencing Methods

Answer 23

• Maxam & Gilbert chemical sequencing
• Sanger chain termination sequencing
• Pyrosequencing
• Next-generation sequencing

Question 24

Sanger – Chain Termination Sequencing

Answer 24

• A modified DNA replication reaction
• chains are terminated by dideoxynucleotides
• The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTP’s and this will terminate extension
  -* Dideoxynucleotides are added separately to each of four tubes proper ratio of dNTP:ddNTP
• DNA polymerase will extend the primer until a ddNTP is encountered
• The chain will end with the incorporation of the ddNTP
• The collection of fragments is a sequencing ladder
• The resulting terminated chains are resolved by electrophoresis
• Fragments from each of the four tubes are placed in four separate gel lanes
• Sequencing gels are read from bottom to top (5′ to 3′)

Question 25

Advances in Sequencing Technology
Dye Terminator Sequencing

Answer 25

• Cycle sequencing is chain termination sequencing performed in a thermal cycler
• fluorescent dye molecules are covalently attached to the dideoxynucleotides, labeling the sequencing ladder at the 3′ ends of the chains
• A distinct dye or “color” is used for each of the four ddNTP’s
• Because the terminating nucleotides can be distinguished by color, all four reactions can be performed in a single tube and resolved in one gel lane or capillary read on an electropherogram

Question 26

Pyrosequencing

Answer 26

• based on the generation of a light signal through the release of pyrophosphate (PPi) upon nucleotide addition:
• PPi is used to generate ATP from adenosine phosphosulfate (APS)
• ATP and luciferase generate light by conversion of luciferin to oxyluciferin
• Each nucleotide is added in turn
• Only one of four will generate a light signal
• The remaining nucleotides are removed enzymatically
• The light signal is recorded on a pyrogram