The advent of DNA profiling technology has greatly improved the rate of solving cases, and its application in paternity testing has deeply affected people's lives and cognition. However, in order to restore the truth of the facts, obtaining accurate DNA identification results with the correct operation is only an important step, and we also need other evidence to corroborate and supplement each other in order to form a complete evidence chain.
But this does not prevent us from first understanding the paternity test from the principle and thinking method, and making theoretical preparations for the interpretation of an appraisal report.
Written by | Qingchao Li (Shandong Normal University)
Paternity testing is an identification technique that uses biological theories and techniques to determine whether there is a parental relationship between individuals. In film and television dramas and daily life, the paternity test we see and hear is often used to identify the "father-son/daughter" relationship, but paternity testing can also identify the "mother-child" relationship, brother-sister relationship and other kinship relationships, the principle is similar, and the reliability varies with the specific situation. Therefore, paternity testing is also commonly used for civil or criminal justice appraisals such as the adoption of abducted and separated children and the identification of the deceased.
"Ancestral chromosomes": the biological basis of paternity testing
The human genome consists of two parts, 23 pairs of chromosomal DNA (nuclear genome) within the nucleus and mitochondrial DNA (mitochondrial genome) within the cytoplasmic mitochondria. The nuclear genome is composed of 23 chromosomes inherited from the parent and the mother, while the mitochondria are all derived from the mother. The origin of the genome constructs the kinship ethics of the entire human society. Paternity ultimately depends on the identification of chromosome sources.
Pictured: The human nuclear genome, containing 23 pairs of chromosomes, one of which is from the father and the other from the mother. When giving birth, a random pair of chromosomes is also chosen to pass on to the child. The last pair of chromosomes is XY for males and XX for females. 丨Source: wikipeida
"Blood Recognition": an ancient method of paternity testing
Paternity testing is not a new problem, people no longer believe in "induction to have children", but instead believe that there must be a cause and a cause, "there must be a son must have a father" has been more than two thousand years. However, the truly scientifically grounded method of paternity testing began to be established in the early twentieth century after the development of the human blood group system and serology. The principle of this identification method is based on serological findings: different human individuals have unique protein antigenicity, and the protein antigenicity tends to be the same between parents and children. For example, the well-known ABO blood group system, if the baby of an O blood type couple is A or B type, then the proven father is not the father; if the baby is an AB blood type, then please call the police 110, the mother is not the mother. However, the repulsive power of this method is limited - detecting A, B, ab blood types all reject the possibility that the baby is biological, but if the baby is O blood, it does not prove that it must be biological.
To solve this problem, HLA detection can be employed. HLA refers to the human leukocyte antigen (Human leukocyte antigen), which has a high polymorphism in the population and is an item that must be detected during organ transplantation, and HLA mismatch can cause rejection. HLA testing is more repulsive to paternity testing, reaching 80%, but requires a large number of blood samples and is not suitable for infants less than six months.
With the deepening of human understanding of the nature of genetic material and the development of DNA detection technology, paternity testing has entered the era of DNA testing. DNA testing became the most accurate method of paternity testing. Earlier DNA detection relied on RFLP (Restriction fragment length polymorphism), or "restriction endonuclear length polymorphism," in which different sequences of DNA were cleaved by restriction enzymes to produce fragments of varying lengths. The most widely used is PCR detection, which mainly identifies paternity by amplifying short tandem repeats (STR) length differences. Children's shoes who love criminal investigation know that contemporary forensic medicine can identify DNA using only traces, relying on PCR amplification. The nucleic acid test of the new crown virus that we introduced earlier also relied on PCR amplification.
Figure: Technological developments in paternity testing in the twentieth century. [1]
What test results are valid?
For a test result to be effective, the experiment needs to be designed according to the experimental principle, and there needs to be a good control in the design: negative control and positive control. According to the principle and experimental operation, the positive result of the positive sample can prove that the testing system can work, and the negative result of the negative sample proves that the testing system is not contaminated. If a positive result is present in a negative control, or a negative result is in a positive control, the test result will be overturned.
In addition, the experiment should be standardized and withstand the necessary repetition, if a sample will be positive and negative, how accurate is it? To sum up, it is theoretically feasible to design reasonable operation specifications. There is a wonderful blood recognition drama in a palace fight drama, but these three are not stained, and there is no scientific reason for blood recognition.
Left: Negative control If a positive result is present, there is a problem with this detection system. Right: The operation should be standardized.
In fact, this is all pediatrics, and there is a story in history that dog blood is not enough, and the blood of people is spilled. Historical records record that Emperor Xiao Yan of Liangwu beheaded Emperor Xiao Baojuan of Southern Qi, seized his palace man Wu Jinghui, and was given the title of Wu Shuyuan. Wu Shuyuan entered the palace in July and gave birth to Xiao Zong, so rumors called Xiao Zongshi the widow of Xiao Baojuan, Xiao Yan did not think so, but became a piece of Xiao Zong's heart disease. In the end, Wu Shuyuan told Xiao Zong the truth: Son, your father is Xiao Baohuan. Xiao Zong was very experimental, planed his father's grave, and dripped his own blood on xue baojuan's remains to recognize his relatives (dripping bone relatives, blood into the bone is biological, there is no scientific reason). Blood seeped in. But there was no positive control for this experiment, so Xiao Zong hacked one of his own sons who had just turned a full moon and got the bones out for further experiments. The blood seeped in, and he really believed that he was Xiao Baojuan's widow. this...... I'd like to say that thankfully no one told him he needed a negative control, as well as a repeat trial.
Exercises after class
What should I do with a negative control for this experiment? Please leave a message for children's shoes to submit answers.
Current methods of paternity testing 1. Detection target: Microsatellite DNA
The testing targets that can be used for paternity testing must meet two elements, stable transmission between parents and children, stable enough to identify their blood relatives, and highly diverse in the population, unique enough to exclude non-blood relatives. In the human genome, there is such a group of DNA fragments, called microsatellite DNA, which can take on this responsibility.
Microsatellite DNA, commonly referred to in animal genetics as "short tandem repeats" (STR), is a type of tandem repeat sequence, consisting of 1 to 6 bases composed of short sequences repeated 5 to 50 times and formed. The human genome contains thousands of STR sequences, and these sequences are often present in non-coding DNA, compared to other DNA sequences, STR mutation rate is higher, and the form of mutation is mainly increased or decreased by the number of short sequence repetitions, not eliminated by natural selection (in layman's terms, unimportant things, the room for variability is large, just like the wheels of a car can only be round, and the shape of the wheel hub is relatively free). Therefore, STR is more polymorphic in the population, known as DNA fingerprinting, and is suitable for use as a paternity test. The identification box of a major brand uses a combination of 15 STR loci plus 1 sex gene.
The number of STR detections and the selection of sites can be optimized according to different ethnic groups and population sizes, generally including thirteen core sites of CODIS. Sex identification genes generally use the tooth enamel protein gene (amelogenin), which has different versions on the X chromosome and on the Y chromosome, called AMELX and AMERLY, respectively, AMELY is six bases shorter than AMELX, so that if amplification yields two long DNA products, it is female (XX), and if one long and one short two amplification products are obtained, it is male (XY).
Figure: Short series of repeating sequences STR. The number of repetitions of short fragments on the same STR site may be different in different human bodies (7 to 11 repetitions in order). The number and sequence of bases for short fragment repeat units at different STR sites are also different (2 to 5 base repeat units in the figure). The size of the DNA product fragment produced after amplification using primers (Primer) increases with the product of SRT repeat units and the number of replicates (the final amplified fragment includes sequences on both sides of the STR in addition to the STR sequence, with a total fragment length in the range of 90 to 370 base pairs). [2]
CODIS core STR site
The CODIS (Combined DNA Index System) DNA Combination Indexing System is a criminal justice DNA database supported by the FBI in the United States, from October 1998 to December 31, 2016, during which there were 13 core STR sites that needed to be registered, and expanded to 20 since January 1, 2017.
Figure: Thirteen core STR sites of CODIS and their positions on chromosomes (AMEL is a sex detection gene)[3]
Figure: The STR site selected for a major kit and its detectable alleles. It contains 13 CODIS core loci and two additional loci of D2S1338 and D19S433, as well as enamel protein sex identification genes. The numerical value of the allele indicates the number of short sequence repetitions of the locus. For example, D8S1179 locus has 8, 9 and other 12 alleles, and the combined probability of the alleles detected by someone amplification is 12x12 = 144 (diploid, so there are two possible alleles per locus), and the possible combinations of each locus can be calculated in turn. The product of all possible combinations of sites is the total number of STR combinations that can be detected in this kit theory.
Figure: The size of all THE DNA fragments detectable by a mainstream kit after STR allele electrophoresis. In actual experiments, all DNA fragments are isolated within a single lane, with short pieces of DNA running fast and long pieces of DNA running slow, so that fragments of DNA of different lengths are separated. Some STR sequence amplifications may have the same amplification, in which case they need to be distinguished by the color of the fluorescent dye attached to the fragment.
2. Detection method: PCR
The current STR detection method mainly uses polymerase chain reaction (PCR) to amplify DNA fragments containing specific STR site sequences and detect their length by electrophoresis to determine the number of STR repetitions. The principle of PCR can be found in "False Positive for Environmental Monitoring, Why Is It Difficult to Eliminate It?" part II of the article.
The kits currently used in mainstream use amplify 15 alleles and 1 sex identification gene in the same tube. The size of the DNA product fragment produced after amplification using primers (Primer) increases with the product of SRT repeat units and the number of replicates (the final amplified fragment contains sequences on both sides of the STR in addition to the STR sequence, with a total fragment length in the range of 90 to 370 base pairs). The product after amplification is isolated and detected using capillary electrophoresis.
Figure: An example of a test result from a major kit. Each STR site can see 1 to 2 peaks, and the number of allele repeats can be judged according to the size. If there are 2 peaks, two different alleles are present at this STR site and one of them should be detected in their children or parents. If it is a peak, the two alleles representing this STR site are the same, and the parents' STR sites should contain this allele, and their children must have this allele.
3. Test sample: THE DNA is stable
From saliva to bones to teeth and hair, nuclear DNA can be found in any nucleated cell in the body. A small amount of DNA can be amplified using PCR technology, which allows a large number of replicates of a small number of TARGETs of STR to achieve the amount of DNA amplification fragments that can be detected. Therefore, as long as the DNA of the object to be tested can be obtained, regardless of the material, it can be used for detection. Usually, paternity testing is similar to the sampling method of covid-19 nucleic acid detection, except that paternity testing only requires scraping a sufficient amount of autologous cells on the inner wall of the mouth.
DNA is stable. For some cases where the subject to be tested has passed away, as long as it is not burned to ash, its hair, bones, and especially the pulp part of the teeth can basically obtain DNA samples that can be used for paternity testing. For hair, follicle-containing samples are far superior to follicle-free samples, but follicle-free hair can also be reported for paternity testing (mitochondrial DNA or DNA extraction and amplification techniques for hair) [4]. If a sample of the deceased's body is not available, samples that can be used for testing can also be obtained on the deceased's clothing or personal items, such as combs. Faced with this situation, it may be necessary to rule out whether the sample has been contaminated with someone else's DNA.
Figure: Chromosomal DNA contained in cells (top right) and samples that can be used for identification, starting counterclockwise from hair, saliva, blood, semen, and urine, the last four samples can take their dried plaques. [5]
The development of modern biotechnology allows noninvasive DNA testing of unborn fetuses. This test is performed by taking the mother's blood through a vein puncture to obtain "cell-free fetal DNA" (cffDNA). The so-called cffDNA is the free circulation of fetal DNA in the mother's blood, derived from the cells of the embryonic trophoblast. cffDNA analysis is a noninvasive prenatal diagnostic method (genetic disease, chromosomal malformation screening, etc.) and is commonly used in pregnant women of advanced age. CFFDNA that appears after nine weeks' gestation can also be used for paternity testing (whether permitted by law requires consultation with the relevant legal person).
4. Instrument Consumables: Similar to nucleic acid testing configuration
The experimental conditions required for paternity testing are basically similar to the configuration of a nucleic acid testing laboratory (refer to "False positive environmental monitoring, why is it difficult to eliminate it?"). Part I), but a better electrophoresis analysis platform is needed.
5. Analysis of results: It may be possible to hammer the tone, or more evidence may be needed
After amplification and electrophoresis, the allele type of the TEST site can be obtained. Compare the allele types between the subjects and you will get the results. Let's look at how to analyze it with the following simplified example.
The first example is an analysis of dna test results from the victim, the crime weapon, and the three suspects. The DNA sample on the murder weapon may come from the victim or from the criminal, and we look at it through the difference in the stripes, and the bands on the murder weapon are different from the victim's sample bands, obviously from people other than the victim. Let's compare the murder weapon and the samples of the three suspects, have you found the suspected murderer?
Picture: Case 1, who is the murderer? [6]
The second example is the case of identifying a biological father (the same practice and principle is used to identify the biological mother). The second column of the baby's DNA test bands originated from the first birth mother and one of the 3, 4, and 5 birth father candidates. For example, the baby's 1st, 4th, and 5th black DNA bands do not exist in the birth mother, this band is inherited from the biological father, and the 2, 3, and 6 black DNA bands are inherited from the birth mother (the birth mother band can be on top). Next, you only need to find the bands that are present in the baby's test results, but do not exist in the birth mother, and which candidate father exists. Did you find it?
Figure: Case 2, who is the biological father? [7]
In the actual identification operation, if there is a paternity relationship between the testees, there is an allelele of each of their STR loci; conversely, if the testee has an allele of the same allele at each STR site, the probability of paternity relationship is more than 99.99% (in practice, the parental index Combined Paternity Index, the calculation and application of CPI, is not discussed in this article, but does not affect the understanding of the topic).
So, if it is difficult to obtain an exact parent sample, can a sibling be identified by paternity testing?
Both men and women can be determined to be siblings of the same mother, or cousins of the same lineage, as long as their mitochondrial DNA is tested and the sequence is consistent (it is not excluded that the table is far away). If it is a brother, the Y chromosome is tested, and the sequence is consistent, it can be determined that it is a half-father brother, or a cousin of the same paternal line (there is no guarantee that the church is closer). As for the other chromosomes, or whether the STR sites match, it is within a probabilistic range. For example, without looking at mitochondrial DNA, a pair of siblings or siblings can theoretically show no blood relationship on chromosomal DNA (the chromosomes selected by sperm and egg gametes are not the same). Theoretically, a couple can also give birth to brothers or sisters with identical chromosomal DNA twice (different ages, but acting like identical twins) (sperm and egg gametes each choose the same chromosome). Although the probability of the above situations is very low (considering that the chromosomes can be recombined during the formation of gametes, the situation is more complicated), the kinship of siblings in the real situation is between the above two extreme cases, so the credibility of the identification results between siblings is uncertain and is not optimal in blood identification.
Therefore, in real life, paternity testing generally needs to be combined with other evidence (parental history, kinship testimony, or the introduction of more blood-related individuals to be identified and analyzed) to make as accurate a judgment as possible.
6. How is the maternal genetic relationship of "chained women" identified?
CCTV News Screenshot: The report's identification conclusion shows that through 60 mtDNA-SNP tests, the tested people are consistent with the maternal genetic relationship. (Source |.) CCTV News Channel)
On February 23, CCTV News broadcast the investigation of the "Feng County Eight-Child Woman" incident, and the Physical Evidence Identification Center of the Ministry of Public Security compared the blood samples of "chain women" Yang Mouxia and Guang Mouying with the biological materials extracted from the relics of Xiaohuamei's mother Pu Mouma, and concluded that "Pu Mouma, Yang Mouxia and Guang Mouying are in line with the biological parent-child relationship". The material presented in the news video shows that the object of this test is mitochondrial DNA (mtDNA).
Mitochondria are organelles located within the cytoplasm that breathe aerobically and produce ATP, the "energy currency." Mitochondrial DNA (mtDNA) is DNA located in the mitochondria and belongs to extranuclear genetic material (DNA is also present in plant chloroplasts). Each mitochondria contains multiple copies of cyclic double-stranded mtDNA, each of which contains a total of 16,569 base pairs, of which there are 37 genes, which are much smaller than the nuclear genome [8]. For animals, the mtDNA in a fertilized egg is mainly inherited from the mother (paternal mitochondria are degraded in the fertilized egg), so the maternal inheritance of mtDNA allows researchers to trace the long-term maternal genealogy and trace the maternal ancestry (the paternal genealogy is performed with the Y chromosome) through mitochondrial DNA.
Figure: Mitochondria are organelles located within the cytoplasm of eukaryotic cells, and their matrix contains circular mitochondrial DNA. (Source |.) wikipedia）
MtDNA mutation rate is higher than nuclear DNA mutation rate, often in the protein coding sequence codon third position mutation, in addition to mtDNA also has two hypervariable control regions (HVR1 and HVR2), so mtDNA contains a relatively rich single-nucleotide polymorphisms (SNP), That is, the types of bases at a single site vary in the population, so the maternal inheritance of the sample to be measured can be analyzed by means of the amount of SNPs contained in the mtDNA. Information about mtDNA was first admissed as evidence by U.S. courts in 1996.
Current mtDNA detection can be done by assaying HVR1 with the highest number of SNPs, assays the full-length sequence of mtDNA, or by scanning selected SNPs throughout the mtDNA genome using microarray chips. The second method provides the most information, while the third method is more suitable for large-scale commercial applications.
It should be pointed out that relying on mtDNA information alone can only determine the maternal genetic relationship between the tested persons, but cannot determine the mother-daughter and parent-sister relationship (the so-called matrilineal genetic relationship between the two people means that in the unknown generation, the two have the same grandmother). The two suspected sisters need to obtain information about their biological mothers through separate identification or, as the bulletin says, introduce more similarly related test reports to better draw conclusions.
Screenshot of CCTV News: Appraisal Opinion (Partial) (Source | CCTV News Channel)
In addition, the appraisal opinion (pictured above) shows that there is a mother-daughter relationship between the suspected birth mother and one of the suspected sisters, Guang Mouying, and if the mother-daughter relationship between the suspected birth mother and the tested person Yang Mouxia (that is, the "chain girl") is added (as explained in the notification), the evidence chain is complete. The assay used in this report is the nuclear genomic STR assay, which detects up to 23 STR sites, which is superior to the kit described in the examples described herein.
epilogue
DNA identification technology has exposed more criminals, corrected more unjust, false and wrongly decided cases, reunited more separated families, and revealed more human tragedies. Professionals should regard professionalism as life, and professional organizations should regard credibility as life. If the world wants the truth, there must be the truth.
bibliography
[1] https://web.archive.org/web/20041119070715/http://www.paternity-answers.com/history-paternity-test.html#1920
[2] https://www.researchgate.net/publication/221912832_DNA_biometrics/figures?lo=1
[3] https://openlab.citytech.cuny.edu/openstax-bio/exam-4/biotechnology-genomics/5/
[4] ishinews.com/no-nuclear-dna-in-rootless-hair-myth-or-fact/
[5] https://openlab.citytech.cuny.edu/openstax-bio/exam-4/biotechnology-genomics/5/
[6] https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/dna-profiling.html
[7] https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/dna-profiling.html
[8] https://en.wikipedia.org/wiki/Mitochondrial_DNA
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