Forensic Biology: Lecture 4 – Separation and Visualization of DNA Molecules

Multiplex PCR results in:

A mixture of DNA molecules of different size (up to 20 or more).

Microvariants

Alleles that are not exact multiples of the basic repeat motif, or sequence variants of the repeat motif—or both.

Electrophoresis

A process where DNA fragments are separated according to size using electrical charges.

Longer fragments travel:

The least (closest to the cathode).

Shorter fragments travel:

The most (closest to the anode).

Smile bands:

Electric field during separation creates heat. Too much heat will create streaking bands or melt the gel matrix and cause the slab to disintegrate.

Agarose

Polysaccharide obtained from seaweed used as supporting medium in gel electrophoresis. Large pore size (200 nm), used for separating larger DNA molecules (ie. RFLP products, single PCR amplicons) 400 bp or greater.

Polyacrylamide

A polymer used in vertical and horizontal electrophoresis; much smaller pores (10-20 nm) separates closely-sized, smaller DNA molecules (i.e., multiplex PCR amplified STRs) that may differ by only a single nucleotide.

Preparation of agarose gel:

• Powder is mixed with electrophoresis buffer (TBE or TAE) and dissolved into solution with heat
• Liquid agarose is poured into a gel slab casting tray and a "comb" is inserted to form wells
• Combs may be square shaped or sharks tooth
• Once the gel has polymerized, the comb is removed and DNA sample is pipetted into the wells

TBE

Tris-borate-EDTA

TAE

Tris-acetate-EDTA

ethylene-di-amine-tetra-acetic acid

EDTA

EtBr

Ethidium bromide; fluorescent visualization of DNA.

DPA

Diphenylamine; colorimetric visualization of DNA.

Native vs Denaturing:

Native = separation of DNA while complementary strands are together (double-stranded)

Denaturing = separation occurs in environment that keeps the DNA single stranded

Capillary electrophoresis advantages:

- Sample injection, separation, and detection can be fully automated
- Samples can be run in an instrument with no technician input
- Uses a fraction of the amount of sample
- Separation takes only minutes
- higher voltages possible because heat is dissipated more rapidly
- Analysis takes place in the instrument - no scanning & photographing of gel required
- Eliminates cross contamination of wells

Capillary electrophoresis disadvantages:

- Prior disadvantage was that samples had to be analyzed sequentially, single capillary cannot process high numbers of samples
- Now units contain capillary arrays that can run multiple samples simultaneously, up to 96 capillaries = high throughput capacity
- Cost: instruments and reagents are very expensive

Ogsten:

small molecules through large pores

Reptation:

large molecules through small pores

False; Electrophoresis is a relative measurement. Position of the DNA on the gel or in the polymer must be compared to a size standard of known DNA fragment size.

True/False: Electrophoresis is an absolute measurement?

Capillary electrophoresis basic components:

- Narrow capillary
- Two buffer vials
- Two electrodes connected to a high voltage power supply
- Laser excitation source
- Fluorescence detector
- Computer for autoinjection and detection

In forensic science, CE is used for:

DNA, gunshot residue analysis, explosive analysis, drug analysis, and pen inks analysis.

Electrophoresis capillaries are:

• Made of glass
• Internal diameter < 50µm - (size of a human hair)
• Contains viscous polymer solution; sieves DNA molecules as they move toward anode
• Similar to gels, the larger DNA fragments encounter more resistance and pass more slowly
• Each capillary is equal to one lane on a gel - can only handle one sample at a time
• DNA samples are mixed prior to injection with an internal size standard to correlate results
• Electric fields are between 10 and 100 times stronger than with gel electrophoresis = faster run times

Performance Optimized Polymers

- dynamically coat the capillary wall to control electroosmotic flow during electrophoresis
- detect alleles differing in size by a single base (up to 250 base pairs in length)
- size same length alleles with a precision of less than 0.15 nucleotide standard deviation
- less than 30 minutes analysis time per sample
- provide capillary life of at least 100 injections
- provide a highly denaturing environment for DNA samples

Electrokinetic Injection

• Electric voltage is applied to the capillary while the end is immersed in the DNA sample
• Flow of the current pulls the negatively charged DNA molecules onto the capillary

Electrophoretic separation of short tandem repeat (STR) fragments is performed at a temperature of:

60°C

Fluorescent dye is incorporated into the amplicons via:

Attachement to the 5' PCR primer or bound to the DNA by intercalation.

Electropherogram

Data output from capillary electrophoresis where fluorescent signals are recorded as graphical peaks.

Fluorophores

Chemical compounds that absorb/emit light of specific wavelengths; can be a dye or protein. Primarily used in DNA labeling are those in the visible region of the spectrum (approximately 400-600nm).

RFU

relative fluorescence units; correlates to relative quantity of DNA in sample based on its response from the detector.

Electopherogram X-axis:

Time

Electopherogram Y-axis:

RFUs

True/False: Time correlates to size of the DNA fragment on the electopherogram:

True; smallest fragments detected first, and the largest fragments detected last.

Molecular extinction co-efficient

ability of dye to absorb light

Quantum yield

efficiency of conversion of absorbed light to emitted light

Photo stability

dye must undergo repeated cycle of excitation without being destroyed

Dye environment

pH, temperature, solvent, and presence of quenchers (ie. hemaglobin)

Fluorescent dye intercalation

Inexpensive but single colored so all molecules must clearly separate by size.

Fluorescently labeled deoxynucleotides (dNTPs)

Both strands labeled in a single dye color.

5' end labeled oligonucleotide primer

- Only single strand is labeled, simplifies interpretation
- Amplicons labeled simultaneously & independently
- Overlapping sizes distinguished by different dyes

ISS

internal size standard; labeled with a different colored dye so that it can be distinguished from the DNA fragments in the sample.

Allelic ladder

tells the instrument what each allele should look like when run through the capillary

Formamide acts as a:

denaturing agent

Teratogen

any factor that can cause a birth defect

Impurities such as formic acid (smaller negatively charged ions), ammonia, and other unknown impurities could:

- Compete with the DNA fragments to reduce the amount of DNA injected
- Produce lower signal-to-noise ratio (lower signal, more noise)
- Degrade and decompose DNA fragments = multiple interference peaks around peaks of interest, reducing resolution and selectivity

High quality formamide should:

- Be greater than 99% pure
- Have a minimum conductivity of 100 µS/cm
- Have low water content
- Be packed under inert gas

True/False: Using water to denature the DNA produces results concordant with those processed with formamide?

True; Water alleviates the risk of working with formamide, but there are drawbacks.

Drawbacks of replacing formamide with water :

- water rapidly evaporates and the DNA forms secondary structures
- DNA stored in water will eventually renature

Formamide is effective in maintaining the DNA in a denatured state even:

- in the presence of stabilizing EDTA (water cannot)
- when left on the instrument at room temperature