DNA separation and detection methods

Question 1

the need for dna separation

Answer 1

pcr reaction in which str alleles are amplified produces a mixture of dna molecules of different sizes (amplicons)
a multiplex can in theory have more than 40 different sized amplicons
all within a narrow size range (about 100-400bp)
resolution needs to be good because there are alleles that differ by a single nucleotide
results need to be reproducible and comparable between different labs

Question 2

basic principles of dna separation

Answer 2

dna is a negatively charged molecule in an aqueous environment
dna migrates from the negative electrode (cathode) to the positive electrode (anode) in electrophoresis
electrophoretic mobility usually determined by a molecule’s charge to mass ratio
however Dan’s charge to mass ratio is 1:1 (1 charge / bp)
so ratio is the same even if the length varies
pcr products of STR is linear
so shape doesn’t affect mobility (protein)
therefore separation is based on the effect of medium (independent var) a d a sieve like-effect on the DNA particles
SIZE

Question 3

electrophoresis

Answer 3

easier for small molecules to migrate through the pores of the gel
larger molecules take longer
separated by size
the higher the voltage, the greater the force felt by the DNA molecules and the faster they migrate

Question 4

slab gel vs CE

Answer 4

slab gel
larger ones at top of gel near wells
smaller at end of gel, migrated further
CE
smaller at the start, signal detected earliest
larges at the end, signal detected latest

Question 5

types of gels

Answer 5

1st gen
agarose
larger pores
used for separating larger pieces of DNA
used in RFLP analysis
used in slab gels

next gen
polyacrylamide
smaller pores
used for separating small (below 500bp) pieces of dna
get high resolution separation
can separate amplicons with 1bp difference,
microvariants 9.3, 10
remember str amplicons range from 100-400bp
can be used in slap or capillary
horizontal or vertical

Question 6

single strand vs double strand

Answer 6

under normal conditions dna will be double stranged
called native or nondenaturing systems
generally better resolution is achieved when dna is single stranded
denaturing system
need to denature prior to separation by CE
use of chemicals such as form amide and urea
these form hydrogen bonds with the bases
increase temperature
a popular technique
dilute sample in 100% formamide
heat sample to denature dna, allows H bonds to form with formamide/urea
“snap cooled on ice”, separates and kept separated

Question 7

capillary electrophoresis

Answer 7

advantages
injection, separation, and detection can be fully automated
can run multiple samples at a time
rapid deparation
heat generated from the electric field is more easily dissipated allowing for use of higher voltages and therefore faster runs
high surface area from tubes of capillaries
excellent sensitivity and resolution
cons,es minute quantity, 1uL/35uL sample
single base pair resolution
less labor intensive during set up
peak info is automatically stored for easy retrieval, allele calls
data is saved
dna can be resampled multiple times

Question 8

components of CE

Answer 8

narrow capillary tube filled with polymer
2 buffer vials
2 electrodes connected to a high voltage power supply
laser excitation source
fluorescent detector
autosampler tray
computer to control sample injection and detection

Question 9

applied bio systems 3500 genetic analyzer

Answer 9

8 capillary system that can easily be upgraded to a 24 capillary system when you’re ready
single line 505nm solid state long-life laser that utilizes a standard power supply and requires no heat-removal ducting
powerful, integrated data collection software and primary analysis software provides real-time assessment of data quality
radio frequency identification RFID technology tracks key consumables data and records administrative info
advanced multiplexing capabilities for DNA fragment analysis with up to 6 unique dyes

Question 10

sample prep

Answer 10

samples are diluted in a solution of deionized formamide
disrupts H bonds and keeps them separate
dilutes salts which aids in electrokinetic injection
an internal size standard is added to each sample
don’t forget to add your positive amp control, your negative amp control, (these are amplified so not difficult to forget at this step) intermittent ladders

Question 11

CE overview

Answer 11

injection
separation
detections

Question 12

CE injection

Answer 12

introducing the DNA sample into the capillary
electrokinetic injection used
capillary is placed in sample along with an electrode
the sample has low ionic strength due to the formamide and high electric field (applied by electrode)
inside the capillary there is a high ionic strength buffer (polymer) and a low electric field
opposites
very small volume used, the rest can be retained for use at a later date
capillary and electrode are placed into the sample solution vial and a voltage is applied
if the sample is ionized and the appropriate voltage polarity is used then the sample ions will migrate into the capillary
this type o injection is known as electrokinietic sampling
because of electric difference and ionic difference

Question 13

sample stacking

Answer 13

concentrates ions in one band
improves resolution and proper sizing
makes for a sharper peak in the electropherogram
like double gels, diff solutions
results when samples are injected from a solution that has a lower ionic strength than the buffer system inside the capillary
when the electric field is applied the resistance and the field strength in the sample increases because there are fewer ions to carry the current in the lower ionic samples
this causes the ions from the sample to migrate rapidly into the capillary
when the ions reach the capillary (buffer has higher ionic strength) they slow down nd stack as a sharp band at the boundary between the sample medium and the elextrophoresis buffer (polymer)

Question 14

CE separation

Answer 14

DNA migrates through the capillary toward the anode
the capillary wall is made of glass or fused silica
36cm in length from injection to detection
silanol groups on the walls of the capillary are negatively charged

Question 15

EOF: Electro-osmotic Flow in CE

Answer 15

silanol groups lining the capillary are negatively charged above pH3, adsorbtion of buffer and cations to the capillary walls creates a double charged layer, when voltage is applied the positively charged ions move in the opposite direction of the DNA migration
causes poor resolution and interferes with DNA migration

Question 16

polymer instead of acrylamide in CE

Answer 16

in CE the same buffers can be used as in gel electrophoresis however instead of a gel matrix (acrylamide or agarose) a polymer serves as the sieving mechanism
linear, flexible chains in solution serve as obstacles for the DNA fragments
instead of gel, very viscous liquid
as with gels smaller more agile fragments move more quickly
polymer is replaced with fresh aliquot in the capillary between runs
EOF is minimized by using POP-4
used as the polymer sieving mechnism inside capillary which coats capillary walls to minimize EOF effects

Question 17

dna fragment sizing and resolution

Answer 17

electrophoresis’s is a relative rather than an absolute techinique
must run size reference standards with known DNA fragment sizes
this calibrates migration times
LIZ size std is included in all samples using a different dye and serves this purpose

Question 18

DNA detection

Answer 18

historically radioactive labels
expensive time consuming and dangerous handling
silver staining
low cost can automate only one eye color and both DNA strands detected so you will see two bands for each allele
can only have as much as you can separate with one dye
2 bands per allele, lowers specificity for alleles that differ by one base pair
fluorescence detection
multicolor analysis
diff fluorophores
rapid and easy to use formats
fluorescent dye is attached to 5’ end of the forward amplicon
other fluorescent techniques exist however this is most common for STR alleles
uses a charge-coupled divide or CCD camera
measured in relative fluorescence units

Question 19

basics of fluorescence

Answer 19

a molecule capable of fluorescence is called a fluorophore
1st step is a photon from a laser source excites a fluorophore electron from its ground energy state S0 to an excited state S’1
2nd step is this electron goes through a conformational change and interacts with its environment resulting in a relaxed excitation state S1
3rd step is a photon (hvem) is emitted at a lower energy when the electron falls back to its ground state
results in a longer wavelength than the excitation photon
stokes shift is the difference between the apex to the absorption and the emission spectra
this allows the use of optical filters to separate excitation light from emission light

Question 20

spectral calibraion

Answer 20

each dye emits its maximum fluorescence at a different wavelengths, but emits in a range or wavelengths just not max
performed to correct for the color overall between the various fluorescent dyes used
inherent to fluorescent dyes
this tells the software how much overlap to expect between colors, and the software will correct
unique to each instrument
affected by environmental conditions
heat wave, most calibrate
not before every run, but change
spectral overlap at specific wavelength can be read by multiple colors
if a new capillary array or any components are exchanged, calibration must run prior to samples
affects interpretation
note: blue and green colors have an especially high degree of overlap and therefore have more “pull-up”
red (ROX) has a max emission at 607 nm and incorporates overlap from blue, green, and yellow, pull up in all 4 colors
the result is if you have very high red peaks, you will most likely have pull up in all dye channels
as compared to very high blue peaks, you will have pull up in green dye channel and maybe yellow, but prob not red