ASCP MB II: Molecular Techniques Part B: Troubleshooting Gel Electrophoresis, PCR, electropherogram

blurry bands / streaking / smearing in agarose gel

- too much DNA (wells overloaded) or excess salt

- desalt samples with a spin column prior to loading to prevent

contamination of reagents with previous PCR products can be reduced by:

incorporating dU into the PCR products and then treating with UNG (uracil-DNA-glycosylase)

you run an assay with an internal control. the control works but one patients internal control does not. What do you do?

repeat the patient

PCR amplification control does not produce a product

Check the original DNA preparation.

- If adequate repeat amplification.

- If not, re-isolate the nucleic acid.

Check DNA concentration

in a PCR reaction, there are two primers with very different melting temperatures and changing locus is not an option. What can one do to maximize amplification?

increase the length of the primer with low Tm by adding a few bases at the 3' end or the 5' end

before reporting results, questionable or inconsistent data must be:

resolved by additional analysis

best way to ensure no contamination or carryover in the lab

use positive displacement pipettes

best way to remove Rnase

bake glassware or tubes at 250C for 4 hrs

bands in the wrong place

- don't heat nucleic acids before running on a native gel

- don't exceed 20 V/cm (measured from anode to cathode, rather than entire gel length) or allow the gel to exceed 30°C.

prevent loading buffer from floating away or sinking to bottom of well

rinse wells with running buffer just before loading

for the sharpest bands

run the gel slowly, at 5 V/cm.

correct amount of DNA

usually a maximum of 100−250 ng/mm well width

magnesium

excess: non-specific binding

too little: reduced yield

low PCR yield

- PCR primer ↑

- DNA template ↑

- Taq polymerase ↑

improve yield / short PCR products in multiplex reaction are weak:

- annealing time and temperature ↓

- extension time and temperature ↓

nonspecific amplification

- ↑ annealing temperature by 2-5 degrees

- ↓ amount of template

- ↓ annealing time

- ↓ number of cycles

short nonspecific products

- KCl concentration ↓

- annealing temperature ↑

- extension time or temp ↑

long nonspecific products

- annealing temperature ↑

- annealing and ext time ↓

- extension time ↓

Guanidium thiocyanate

nucleic acid protector; stabilize double stranded DNA

Dimethylsulfoxide (DMSO)

used to destabilize template secondary structure

peak 2x as high

repeated nucleotide.

ex: GTTAC

used to keep DNA/RNA in denatured state during analysis

formamide in sample, urea in gel, and/or ht denaturing electrophoresis

EDTA

binds Mg in DNA storage buffer

long probes (500-5000 bp)

- greater specificity

- decreased background

- less affected by point mutation/polymorphism

- slower hybridization rates

short probes (<500 bp)

- less specific

- possibility of random repeat in genome sequence

- more sensitive to point mutation/polymorphism

melting temperature

- 4°C(# G/C nucleotides) + 2°C(# A/T nucleotides)

- 50% of DNA is single stranded

copies of DNA after n cycles

2^n

agarose gel

- concentration 0.8-2.5%

- best for larger fragments (200-2000bp)

polyacrylamide gel

- concentration 3.5-20%

- best for smaller fragments(<1000bp) and ssDNA

hot start PCR

prevents low-temp mispriming; reduces the presence of undesired products and primer dimers due to non-specific DNA amplification.

denature, anneal, extend (approx temperatures)

90, 50, 70

hybridization temperature

usually 5 degrees below probe Tm

threshold cycle (CT)

PCR cycle at which sample fluorescence crosses the fluorescence threshold

more specificity

- Incorporate DMSO or formamide into the PCR reaction

- Use a high fidelity polymerase

- Use nested PCR

blank

-Controls for contamination

- Contains all reagents except DNA template

Negative control

- Controls for specificity of the amplification reaction

- Contains all reagents and a DNA template lacking the target sequence

single band

undigested high-quality DNA

TAE (Tris-Acetate EDTA)

- better conductivity, faster migration, better resolution of small fragments

TBE (Tris-Borate EDTA)

- better resolution of larger fragments, better buffering capacity

primer design

- GC content should be approx 50%

- opt length is 18-24 bp

- forward and reverse should have similar Tm so both will hybridize optimally at same annealing temp

- avoid hairpins, dimers, or self-dimers

GC

- higher content = ↑ Tm and annealing temp

- bonds are less specific

annealing temperature

should be 5 degrees lower than Tm so most primers bind

Quantify amount of DNA extracted from a blood specimen diluted 1:30. Absorbance reading @ 260 nm is 2.545. If absorbance @ 280 nm is 1.406, and if DNA extract was resuspended in 0.800 mL of EDTA, DNA yield is:

2.545 Abs x 50 (ug/mL) x 30 = 3,817.5 ug/mL