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7BBPM027: Forensic Genetics > DNA Amplification and detection with Capillary Electrophoresis > Flashcards

DNA Amplification and detection with Capillary Electrophoresis Flashcards

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1
Q

How is DNA evidence processed?

A
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2
Q

What are Short tandem repeats (STRs)?

A
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3
Q

How is the primer designed for STR amplification?

A
  • Primers specific to location
  • Consider CG content and effects annealing temperature
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4
Q

How do we detect and differentiate between out PCR products- especially if we have 16 STR markers?

A

o Use **capillary electrophoresis (CE) **to separate DNA amplicons based on size
o Multiple fluorescent dyes used to detect amplicons (incorporated into PCR primers)
o Increase in number of markers targeted means systems need to be able to detect up to 6 dyes
 **DNA17 uses 5 dyes **

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5
Q

What is the AmpFlSTRTM NGM SelectTM express kit designed for and what does it use?

A
  • Specifically designed for forensic or paternity use
  • 17 loci (DNA17)
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6
Q

What are the alleles of DNA17?

A

Amelogenin
D3S1358
vWA
D16S539
D2S1338
D8S1179
D21S11
D18S51
D19S433
TH01
FGA
D10S1248
D2S441
D1S1656
D12S391
And the highly polymorphic SE33

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7
Q

Name 5 common dyes used for AmpFlSTRTM NGM SelectTM express kit

A
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8
Q

How does capillary electrophoresis work?

A
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9
Q

What are the conditions for STR typing and what is each of the conditions?

A

* Spectral resolution
o Need to separate different fluorescent dye colours

*** Spatial separation **
o Need to be able to separate alleles that differ in size by a single nucleotide

* Sizing precision
o Need to ensure that between runs sizing is consistent so that alleles can be properly called

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10
Q

Tell me about spectral resolution

A
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11
Q

Tell me the amplicon structure when it comes to spectral resolution

A
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12
Q

Tell me about spatial separation

A
  • 5 dye colours are not enough to separate 17 markers and provide a calibration standard
    o One dye must be allocated to the** internal size standard**
  • Amplicons are separated by varying their size
  • All alleles are one locus must be distinguishable from all alleles of another locus assigned to that dye colour
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13
Q

With spatial separation, the alleles must be distinguishable from all alleles of another locus. whats needed and how can this be done?

A

**Required manipulation of amplicon size with: **
* **Primer design- **design primers with closer to further away from repeat region
* Mobility modifiers-makes heavier so moves at different rate to other one

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14
Q

Amplicon size: primer design

A
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15
Q

With amplicon size in spatial separation, where can primers go and what is the size of the fragment determined by ?

A
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16
Q

Tell me about mobility modifiers
Whats their structure?

A

They provide larger DNA amplicons without the need to actually amplify the DNA equivalent to its length, thereby reducing the extent of the amplification

Mobility-modifiers [19] were introduced by Applied Biosystems (Foster City, USA) in 2001 to provide larger DNA amplicons without the need to actually amplify the DNA equivalent to its length, thereby reducing the extent of the amplification. By incorporating hexaethylenoxide (HEO) units into a primer the mobility-modifying polymers can provide larger DNA amplicons. Since the mobility-modifying polymer is not amplifiable, to detect the increased amplicon size the polymer must be linked to a fluorescent dye in order to detect the DNA strand where the mobility modifier is incorporated. This limits the length of this polymer to only short sizes to avoid any overlapping with the labelled PCR products present in the electropherogram. .

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17
Q

What must spatial separation be able to do?

A
  • Must be able to resolve alleles that differ by 1 base pair
    o E.g., TH01 alleles 9.3 and 10
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18
Q

With spatial separation, what does the separation need to be over the range of?

A

100-500bp

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19
Q

How is electrophoresis used in spatial separation?

A

 Phosphate group carries a negative charge and molecules move towards the anode (positive electrode)
 Speed of migration of DNA fragments through capillary is dependent on fragment size

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20
Q

Tell me multiplexing using size and dyes

A
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21
Q

What is gel electrophoresis and the two types of gels which can be used?

A
  • Flat (slab) gel immersed in a tank of electrophoresis buffer
  • Electric current flows through the tank
    o DNA fragments migrate through gel

Agarose Gel and Polyacrylamide gel

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22
Q

What type of gel is suitable for STR analysis and why?

A

* Agarose gel:
o Resolution not high enough for STR analysis (roughly 10bp)

* Polyacrylamide gel:
o Pore size of 100-200Å- good for STRs as able to differentiate by 1bp

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23
Q

What are the two main components of capillary electrophoresis (CE)?

A

Glass capillary (fused silica)
Electrokinetic injection

24
Q

Tell me about the glass capillary in CE

A

**Glass capillary (fused silica) **
o Filled with polymer
o 50-100µm diameter
o 100-230cm in length
o 1-96 capillaries for simultaneous analysis
o Each capillary equivalent to lane on slab gel

25
Q

Tell me about the electrokinetic injection in CE

A

Voltage applied to sample then pulls negatively charged DNA molecules into capillary

26
Q

Draw the capillary electrophoresis schematic

A
27
Q

Tell me how the separation of the DNA fragments works with capillary electrophoresis

A
  • Electric current pulls -ve charge DNA towards anode
  • DNA fragments are separated by size:
    o Polymer within capillary provides effective ‘pores’
    o Smaller molecules can move more easily through the pores
    o Therefore, the smaller DNA fragments migrate through the capillary faster than the larger fragments
    ** Separated based on size **
28
Q

Why is a “Post PCR” work area needed?

A
29
Q

What is the plate set up for CE?

A
30
Q

What is an Electropherogram ?

A

Fluorescent intensity plotted against detection time

31
Q

What do the peaks represent in a Electropherogram?

A

**Peaks represent amplified fragments **
o Each peak corresponds to a different sized fragment and allele

  • One big peak can assume is homozygous individual
32
Q

In a Electropherogram what is run along with the samples and why?

A
  • Size standard run alongside samples to measure and keep consistent the migration through the capillary (shown in orange to the right)
  • E.g., if size standard was 100 (fragment size) and peak on profile at same point then know the fragment is the same size
33
Q

What is run with each sample in the CE and what is this?

A

Internal size standard (run with each sample)
* Specific DNA fragments of known size which are defined and used to size unknown fragments

34
Q

What is only run once per injection i.e., 16 wells for our experiment, what is this?

A

Allelic ladder (run once per injection, 16 wells)
* Comprised of DNA fragments that represent common alleles at a locus- specific to kit e.g., NGM select express

35
Q

Interpretation of the electropherogram from the CE

A
36
Q

Casework electropherogram

A
37
Q

CE troubleshooting

A

* Bubbles in capillary or gel. want to avoid because this disrupts the migration of the DNA and therefore doesn’t run as uniformly as would like
* Signs of capillary failure- gradual loss of resolution, high baseline, noisy data, or peaks under peaks throughout the sequence, shoulders on peaks or irregular peaks
* Mobility of the sample can be affected by the run conditions: buffer type, concentration, and pH, run temperature, the voltage applied, and the type of polymer used

38
Q

What are shutter peaks in electropherograms?

A

Stutter peaks are found in almost every electropherogram. Stutter peaks are small peaks that occur immediately before or after a real peak. During the PCR amplification process, the polymerase can lose its place when copying a strand of DNA, usually slipping forwards or backwards four base pairs. The result is a small number of DNA fragment copies that are either one repeat larger or smaller than the true fragment being amplified.

39
Q

DNA profiling involves the PCR amplification step. PCR uses the DNA polymerase enzyme to help with elongation. What issues can this come into when undertaking PCR and explain the issues which can occur due to this?

A
40
Q

What are Microvariant and “off ladder” alleles ?

A

* Microvariant alleles –> alleles that are not exact multiples of the basic repeat motif or sequence variants of the repeat motif or both
*** “Off ladder” alleles –> **rare alleles that are not included in the allelic ladder

41
Q

What are Tri-allelic patterns?

A
42
Q

What are Null alleles or “drop out” ?

A
43
Q

What is fronting and tailing?

A
44
Q

Whats spectral pull-up?

A
45
Q

Whats degradation or the ski-slope effect?

A
46
Q

What are some problems that electropherograms can run into?

A
  • Shutter peaks
  • Incomplete adenylation
  • Tri-allelic patterns
  • Null alleles or “drop out”
  • Fronting and tailing
  • Spectral Pull-up
  • Degradation- ski-slope effect
47
Q

What are the artifacts in the electropherogram?

A
48
Q

Describe the“Goldilocks effects

A
  • In complement adenylation can be due to too much template DNA which means that there is not enough nucleotide to ensure amplicons are adenylated
  • Too much and Too little DNA shown above
  • Hence why 0.5-1ng of DNA is best
  • If peaks are too low then we can modify the amount of DNA that goes through capillary which can be done via the electrokinetic injection
49
Q

If there is too much or too little DNA then what can be done to fix this problem?

A

**Modifying electrokinetic injection **
* Control the fluorescent signal by working out the amount of DNA
* The injection parameters: reduce or increase signal strength and improve resolution
* Aim: to inject sufficient DNA to yield peaks of adequate height while maintaining resolution and read length

50
Q

Why is it necessary to have a pre and post PCR areas?

A

Avoid contamination of PCR amplified products getting into pre-PCR reactions

51
Q

What is adenylation?

A

Addition of extra adenine on chain at end of PCR reaction

52
Q

Who do we add formamide to the PCR product before loading onto the 3130 genetic analysers?

A

Prevent single strands turning double stranded

53
Q

In lay terms, what is an allelic ladder?

A

A reference set of alleles

54
Q

In lay terms what is a size standard?

A

The size of the DNA fragment compared to the size allele its matched to

55
Q

What can be altered on the CE to improve the DNA profile obtained?

A

The electrokinetic injection time and the amount of DNA put through the capillary

7BBPM027: Forensic Genetics (16 decks)

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