Resolution and Detection Nucleic Acidscsivc.csi.cuny.edu/ChangHui.Shen/files/Ch5.pdf · Nucleic Acids Chapter 5. Molecular Diagnostics Fundamentals, Methods and Clinical Applications

Molecular Diagnostics Fundamentals, Methods and Clinical ApplicationsSecond Edition

Copyright © 2012 F.A. Davis Company

Resolution and Detection of Nucleic Acids

Chapter 5



Objectives Explain the principle and performance of electrophoresis as it

applies to nucleic acids. Compare and contrast agarose and polyacrylamide gel

polymers. Explain the principle and performance of capillary

electrophoresis as it is applies to nucleic acid separation. Describe the general types of equipment used for

electrophoresis. Discuss methods and applications of pulsed field gel

electrophoresis. Compare and contrast detection systems used in nucleic acid

applications.



Gel Electrophoresis

Electrophoresis is the movement of molecules by an electric current.

Nucleic acid moves from a negative to a positive pole.



Gel Electrophoresis

When DNA is applied to a macromolecular cage or gel such as agarose or polyacrylamide, its migration under the pull of the current is impeded.



The movement of molecules is impeded in the gel so that molecules will collect or form a band according to their speed of migration.

500 bp

200 bp

50 bp

% agarose: 2% 4% 5%

500 bp

200 bp

50 bp

500 bp

200 bp

50 bp

The concentration of gel/buffer will affect the resolution of fragments of different size ranges.



Gel Electrophoresis

Slab gel electrophoresis can have either a horizontal or vertical format.

Sample is introduced into wells at the top of the gel. Because each nucleotide has one negative charge, the charge‐

to‐mass ratio of molecules of different sizes will remain constant. DNA fragments will therefore migrate at speeds inversely related to their size.



Very large DNA molecules are separated by pulsed field gel electrophoresis (PFGE).

http://www.bio.davidson.edu/courses/genomics/method/pulse_field.html

Very large pieces (50,000–250,000+ bp) of DNA cannot be resolved efficiently by simple agarose electrophoresis. Even in the lowest concentrations of agarose, megabase fragments are too severely impeded for correct resolution.

Pulses of current applied to the gel in alternating dimensions to enhance migration.



Types of PFGE Field inversion gel

electrophoresis (FIGE): alternating positive and negative poles In this type of separation, the

DNA goes periodically forward and backward. FIGE requires temperature control and a switching mechanism.

Transverse alternative field electrophoresis (TAFE): transverse‐angle reorientation of poles on a vertical gel

Contour‐clamped homogenous electric field (CHEF): alternating polarity in an electrode array

Rotating gel electrophoresis (RGE): rotating gel with fixed poles

http://www.nal.usda.gov/pgdic/Probe/v2n3/puls.html



Application Bacterial typing for epidemiological purposes ‐ require the resolution of chromosome‐sized fragments of DNA. Enzymatic digestion of genomic DNA will yield a set of fragments that produce a band pattern specific to each type of organism. By comparing band patterns, the similarity of organisms isolated from various sources can be assessed. This information is especially useful in determining the epidemiology of infectious diseases, for example, identifying whether two biochemically identical isolates have a common source.



Polyacrylamide Gel Electrophoresis (PAGE)

Acrylamide, in combination with a cross‐linker, methylene bis‐acrylamide

Synthetic, consistent polymer

Polymerization catalysts: ammonium persulfate (APS) plus N,N,N',N'‐tetramethylethylenediamine (TEMED), or light activation APS produces free oxygen radicals in the presence of TEMED to drive the

polymerization mechanism. Excess oxygen inhibits the polymerization process. Therefore, deaeriation, or the removal of air, of the gel solution is done before the addition of the nucleating agents (APS & TEMED).

Resolves 1 bp difference in a 1 kb molecule (0.1% difference)



composition of polyacrylamide gels The composition of polyacrylamide gels is represented as the total

percentage concentration (w/v) of monomer (acrylamide with cross‐linker), T, and the percentage of monomer that is cross‐linker, C.

For example, a 6% 19:1 acrylamide:bis gel has a T value of 6% and a C value of 1/20, or 5%.

Increasing T decreases the pore size proportionally. The minimum pore size (highest resolution for small molecules) occurs at a C value of 5%. Variation of C above or below 5% will increase pore size. Usually, C is set at 3.3% (29:1 ) for native and 5% (1 9:1 ) for standard DNA and RNA gels.



Advantage Higher resolution capability of

polyacrylamide for small fragments.

The components of polyacrylamide gels are synthetic; thus, there is not as much difference in batches obtained from different sources.

Altering T and C in a polyacrylamide gel can change the pore size and, therefore, the sieving properties in a predictable and reproducible manner.



Capillary Electrophoresis (CE) Separate organic chemicals

(pharmaceuticals and carbohydrates) and inorganic anions and metal ions.

Separates solutes by charge/mass ratio. Negatively charged molecules

are completely ionized at highpH, whereas positively charged solutes are completely protonated in low pH buffers.

Faster and cheaper than HPLC

Capillary gel electrophoresis is used to separate nucleic acids.

=+ + +

+

++

- -

=



Capillary Gel Electrophoresis (CGE) Thin glass capillary 30–100 cm x 25–100

m internal diameter ‐ fused silica ‐ allowing for the passage of

fluorescent light

Linear or cross‐linked polyacrylamide or other linear polymers used for sieving

Separation based on size

More rapid, automated than slab gels Run at higher charge per unit area Electro‐kinetic injection of sample

Capillary electrophoresis separates particles by size (small, fast migration; large, slow migration) and charge (negative, fast migration; positive, slow migration).



Electrophoresis Buffers Buffers carry current and protect samples during electrophoresis.

Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE), and Tris Phosphate EDTA (TPE) are used most often for DNA. TBE has a greater buffering capacity and will give sharper resolution than

TAE buffer. TBE is generally more expensive than TAE, and inhibits DNA ligase which may cause problems if subsequent DNA purification and ligation steps are intended.

10 mM sodium phosphate or MOPS buffer is used for RNA.

Buffer additives modify sample molecules. Formamide, urea (denaturing agents)



Electrophoresis Equipment

Horizontal or submarine gel



Electrophoresis Equipment

Vertical gel



Electrophoresis EquipmentCombs are used to put wells in the cast gel for sample loading.

Regular comb: wells separated by an “ear” of gel

Houndstooth comb: wells immediately adjacent



Gel Electrophoresis Requirements

Use the proper gel concentration for sample size range. 0.5%–5% agarose 3.5%–20% polyacrylamide

Use the proper comb (well) and gel size.



Sample LoadingLoad sample mixed with tracking dye (dye + density agent).

Tracking Dye ComigrationBromophenol Xylene Cyanol

Gel % Blue (Nucleotides) (Nucleotides)Agarose0.5–1.5 300–500 4000–50002.0–3.0 80–120 700–8004.0–5.0 20–30 100–200

PAGE4 95 4506 60 2408 45 16010 35 12012 20 7020 12 45



Detection

Detect bands by staining during or after electrophoresis.

Ethidium bromide: for double‐stranded DNA

SyBr green or SyBr gold: for single‐ or double‐stranded DNA or for RNA

Silver stain: more sensitive for single‐ or double‐stranded DNA or for RNA and proteins



Summary Electrophoresis is used to separate molecules by size and/or charge.

Nucleic acid fragments can be resolved on agarose of polyacrylamide gels.

PFGE is used to resolve very large DNA fragments. CGE is more rapid and automated than slab gel electrophoresis.

The choice of electrophoresis method depends on the type and size of sample.

Documents

Resolution and Detection Nucleic Acidscsivc.csi.cuny.edu/ChangHui.Shen/files/Ch5.pdf · Nucleic Acids Chapter 5. Molecular Diagnostics Fundamentals, Methods and Clinical Applications