Transcript fourth quarter atlas analysis

许冰莹,
昆明医科大学法医学院
Tel:0871-65922837；13769175605
Email:[email protected]
Fourth quarter: atlas analysis
Information is tied together with multiplex PCR and data analysis
AmpFlSTR® Identifiler™ (Applied Biosystems)
D8S1179
TH01
D3S1358
VWA
D19S433
AMEL
D21S11
D5S818
D7S820
D13S317
TPOX
CSF1PO
D16S539
D2S1338
D18S51
FGA
1 integrated analysis vs.
16 separate runs
In STR analysis practice, not every batch of every
sample analysis, can be completed accurately
and automatically by computer program
• Sometimes sample automatic
classification result is wrong, need
professional personnel adjustment
parameter analysis, to analysis to obtain
the correct classification
Affect the classification
• 一、Classification standard allelic naming
errors
• 二、Within the logo don't mistake
• 三、Base jumping or swing
• 四、stutter peak
• 五、A template depends on A
Affect the classification
•
•
•
•
•
六、Other pseudo peak
七、Gender recognition graph anomalies
八、Peak of the balance
九、Heterozygosity lost or 3 alleles
十、off-ladder peak
一、Classification standard allelic
naming errors
Ladder, each each loci alleles are not completely
equal amounts. After electrophoresis detection,
the signal strength is different also
If a loci of the a allele fragments: Volume is
relatively small, the sample quantity is less,
and the peak threshold is set too high
All the alleles are reduce a repeating unit
1、If a particular loci alleles first fragment: Appear
before small a repeating unit of pseudo peak (copy
slip product), sample quantity is big, and the peak
on the threshold value is set too low
All the alleles increase a repeating unit name
replication slippage ：The main cause of
STR polymorphisms
Action Taken
• 1、Synchronous analysis of known
reference samples （9947A）
• 2、Check whether the loci alleles named
Ladder is accurate.
二、Within the logo don't mistake
Identify the correct internal standard
solve
1、Signal is too low
Adjust peak threshold
solve
Increase in scalar electrophoresis again
1、Analysis of range error
解决
To adjust the scope of analysis
200
300
350 400
340
3.Electrophoresis data is not
complete
Internal standard fragment is not all of
them Collection, internal standard larger
pieces missing
More than 400 bp fragment don't read
Electrophoresis again
cause
• 1、Environmental temperature has
significant effects on speed of
electrophoresis
• 2、Electrode buffer, capillary, etc. Use too
much times, Electrophoresis resolution
decreased
三、Base jumping or swing
• Base jumping or swing ：Baseline is too high,
more than peak threshold
•
Countless fragment peaks
cause
• 1、Call matrix file error or are not suitable
for analysis
• 2、Peak signal is too strong, jamming
signals could not be eliminate completely
take measures
• Avoid blindly increase the sample amount:
• Peak signal is too strong
Reduce the sample quantity After dilution
products, electrophoresis again
• The ideal peak charts ：1000-3000
四、stutter peak
• In STR classification mapping, often
before a target STR allele peak, there is
little the position of a repeating unit a weak
signal peak
This extra peak is due to the PCR amplification, fragments
amplified copy slip form, called the shadow zone (stutter
band) or shadow peak stutter (peak)
.
• Stutter peak height or peak area should not
exceed 15% of the purpose of allele peak
• Always in front of the objective allele peak small
one repeating unit
• Within the same loci, long alleles product
relatively short allele stutter
• Peak signal is too strong and homozygous
genes, are more likely to stutter peak was
observed
take measures
• Augment
To reduce the amount of
DNA samples
• electrophoresis
Reduce the sample
quantity, reduces the peak signal strength
• Mention peak threshold
• Adopting peak signal filtering
• With higher thermal stability of DNA
polymerase activity increases
Affect the mixed samples
五、A template depends on A
• Still have A kind of phenomenon, in the
PCR process is dependence on
amplification products of template to add A
• DNA polymerase can, in the end of the
amplification products of 3 '- nonspecific to
connect A adenosine A
A-----------------------------------A
Through the primer design and the amplification
cycle after the end of heat preservation, can make
each fragment amplification products 3 '- are at the
end add A A
预变性
95℃,11min
变性
95˚C,1min
35
60℃,
60min
延伸
72˚C, 1min
退火
59˚C, 1min
• Each allele fragments of all amplified
product bases of DNA molecules are
exactly the same. Electrophoresis in single
piece
• If the achieved only half of A process, can
make the same allele fragments
amplification contains added A and not A,
two DNA fragments, which is A base.
• The a allele two amplification product in
electrophoresis, were identified as two
peaks. Two peak close to, in the base into
a peak, fork pointed part formed two peaks,
referred to as a double peak.
• Add A product peak fragment size and
corresponding allelic ladder Genes in
same size, was named digital
• Without A product, no corresponding allelic
ladder, was identified as off - ladder
cause
• 1、When amplification DNA template
• 2、Amplification system, with PCR
inhibitor
• 3、Taq polymerase or severe attenuation
due to less quantity.
attention
• 1、Double peak phenomenon in small
fragments alleles than large fragments, etc
A gene is more common.
• 2、If on a sample of data, in a larger
pieces Fixed alleles in a single peak, then
to consider Samples in the loci alleles
micro variations may occur
六、Other pseudo peak
• In the electrophoresis process, when
equipment appear short pulse output
current, peak figure will appear in the data
signal baseline suddenly beating.
• Peak in the figure is shown as figure is too
high the fault type of peak suddenly, the
pseudo peak may be identified as the
fragment peaks
• "Wedge" peak
cause
• Buffer containing solid particles, the
particle surface if the negatively charged
•
Stick the peak charts
• With d = 0.25 um filter, water filter for
compound electrode buffer
七、Gender recognition graph
anomalies
• At present the commonly used commercial
kits
• AmpFlSTR® Identifiler
Amelogenin
• AmpFlSTR® SinofilerTM
（性别STR基因
• PowerPlex ® 16
座）
“15+1”STRGene Locus
• For most individuals ：Amelogenin
detection, can accurately identify the sex
of the individual.
Abnormal one
• Male individual Y chromosome
AmelogeninRare is missing
expression
• Don't have access to the Y chromosome
amplification products
result
• Men samples were wrong that is female
solve
• Y-STRamplification
Exception 2
• Male individual X chromosome amelogenin
Rare is missing
expression
• Only Y chromosome amplification products
result
• Men's samples didn't detect the X
chromosome
solve
• X-chromosome Amelogenin Primer
amplification
八、Peak of the balance
• A loci of two segments of heterozygote
peak signal strength differences of the
phenomenon is more common, the main
reason is that amplify imbalance
• Electric sample, small fragments faster
into the capillary Small pieces was slightly
higher than the peak peak larger pieces.
• Typically, small fragments peak area is bigger
than big fragment peaks.
• However, peak area should be larger pieces in a
small segment of the peak area More than 70%.
That is the same different loci alleles The peak
area of difference should be within 30%.
• If the two alleles of peak height difference is
more than 70%, you will need to consider may
be mixed samples
cause
• 1、Amplification DNA template is too little
• 2、DNA Template degradation
• 3、, exist in the PCR reaction system
inhibitors: hemoglobin and its derivatives,
etc
Loss of heterozygosity
Tumor samples
• 1、Loss of heterozygosity
• Heterozygous samples in the
corresponding loci detected one allele,
similar to homozygous test results.
• But in peak area values and signal
strength and other heterozygote
heterozygote peak area of sample.
九、Third-class a gene
Cause:
The extra chromosome; Primer combination point mutations
• Samples should be at the check out 3 or
multiple genes 4 allele
And pollution is more than often affected with
batches of samples.
十、Off-Ladder峰
1 LIZIdentification error
、
solve
To adjust the scope of analysis
200
300
350 400
340
2、Laddernaming errors
3
Mutant allele
、
The influencing factors of spectrum
analysis
• sample
• electrophoresis
• external environment