fourth quarter: atlas analysis

钟树荣（ PhD ，副教授）

昆明医科大学法医学院

AMEL

D3S1358TH01

TPOX

D2S1338

D19S433

FGA

D21S11

D18S51

CSF1PO

D16S539

D7S820

D13S317

D5S818

VWA

D8S1179

1 integrated analysis vs. 16 separate runs1 integrated analysis vs. 16 separate runs

Information is tied together with multiplex PCR and data analysis

AmpFlSTR® Identifiler™ (Applied Biosystems)

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

1、 Signal is too low Adjust peak threshold

Increase in scalar electrophoresis again

solve

solve

1、 Analysis of range error

To adjust the scope of analysis

200 300

340

350 400

解决

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

60 ,℃

60min

延伸72˚C, 1min

退火59˚C, 1min

35

• 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

• AmpFlSTR® SinofilerTM

• PowerPlex ® 16

Amelogenin（性别 STR 基因座）

“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

• Don't have access to the Y chromosome amplification products

• Men samples were wrong that is female

• Y-STRamplification

expression

result

solve

Exception 2

• Male individual X chromosome amelogenin Rare is missing

• Only Y chromosome amplification products

• Men's samples didn't detect the X chromosome

• X-chromosome Amelogenin Primer amplification

expression

result

solve

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

• 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.

Tumor samples

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

、LIZ

Identification

error

To adjust the scope of analysis

200 300

340

350 400

solve

2 、 Laddernaming errors

3

、Mu

tant

allele

The influencing factors of spectrum analysis

• sample

• electrophoresis

• external environment