How is maternal genetic relationship determined? | The scientific principle of DNA paternity testing

The advent of DNA profiling has greatly improved the crime-solving rate, and its application in parent-child relationship identification has profoundly affected people's lives and cognition. However, to restore the truth of the facts, using correct operations to obtain accurate DNA identification results is only an important step. We also need other evidence to corroborate and supplement each other to form a complete chain of evidence.

But this does not prevent us from first understanding paternity testing from the perspective of principles and ideas, and making theoretical preparations for interpreting an identification report.

Written by Li Qingchao (Shandong Normal University)

Paternity testing is an identification technology that uses biological theories and techniques to determine whether there is a biological relationship between individuals. In movies and TV shows and in daily life, we see and hear that paternity testing is often used to identify the "father-son/daughter" relationship, but paternity testing can also identify the "mother-child" relationship, siblings and other relatives. The principle is similar, and the reliability varies with the specific situation. Therefore, paternity testing is also often used in civil or criminal judicial identification such as the recognition of abducted and lost 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 chromosome DNA in the cell nucleus (nuclear genome), and mitochondrial DNA in the mitochondria of the cytoplasm (mitochondrial genome). The nuclear genome is composed of 23 chromosomes inherited from the father and mother, while mitochondria are all from the mother. The source of the genome has built the blood ethics of the entire human society. The parent-child relationship ultimately depends on the identification of the source of the chromosomes.

Figure: The human nuclear genome contains 23 pairs of chromosomes, one of each pair comes from the father and the other from the mother. When giving birth, one of each pair of chromosomes is randomly selected to pass on to the child. The last pair of chromosomes for males is XY, and for females is XX. 丨Source: wikipeida

“Blood test”: an ancient method of paternity testing

Paternity testing is not a new issue. It has been more than 2,000 years since people stopped believing in "induction of children" and started to believe that there must be a cause for every effect and "like son, like father". However, the real scientifically based paternity testing method was established only after the development of the human blood type system and serology in the early twentieth century. The principle of this testing method is based on the results of serological tests: different human individuals have unique protein antigenicity, while the protein antigenicity between parents and children tends to be the same. For example, in the well-known ABO blood type system, if the baby of a couple with type O blood is type A or B, then it is proved that the father is not the biological father; if the baby is type AB, then please call 110 to report that the mother is not the biological mother. However, the exclusion power of this method is limited - testing A, B, and AB blood types all exclude the possibility that the baby is a biological child, but if the baby is type O blood, it cannot prove that it is definitely a biological child.

To solve this problem, HLA testing can be used. HLA refers to human leukocyte antigen, which has a high degree of polymorphism in the population and is a must-test item during organ transplantation. HLA mismatch will cause rejection. HLA testing for paternity testing has a strong rejection rate of 80%, but it requires a large amount of blood samples and is not suitable for infants under six months old.

As human beings' understanding of the nature of genetic material continues to deepen and the technology of DNA detection develops, paternity testing has entered the era of DNA testing. DNA testing has become the most accurate method for paternity testing. Earlier DNA testing relied on RFLP (Restriction fragment length polymorphism), which is the principle that DNA of different sequences will produce fragments of different lengths after being cut by restriction endonucleases. The most widely used test is PCR testing, which mainly identifies parent-child relationships by amplifying the length differences of short tandem repeats (STR). Those who are interested in criminal investigation know that contemporary forensic medicine can identify DNA using only trace traces, relying on PCR amplification. The nucleic acid test for the new coronavirus that we introduced earlier also relies on PCR amplification.

Figure: The development of paternity testing technology in the 20th century. [1]

What kind of test results are valid?

For a test result to be valid, the experiment needs to be designed according to the experimental principle, and there needs to be good controls in the design: negative control and positive control. According to the principle and experimental operation, a positive result in a positive sample can prove that the test system can work, and a negative result in a negative sample can prove that the test system is not contaminated. If a negative control has a positive result, or a positive control has a negative result, the test result will be overturned.

In addition, the experiment must be standardized and able to withstand necessary repetitions. If a sample is positive sometimes and negative sometimes, how can it be accurate? In summary, it is ① feasible theory ② reasonable design ③ standardized operation. There is a wonderful blood test in a palace drama, but none of these three conditions are met. There is no scientific reason for blood test.

Left: If a positive result appears in the negative control, there is something wrong with the detection system. Right: The operation should be standardized.

In fact, these are all child's play. There is also a story of blood recognition in history that is not enough. It is about blood dripping to recognize relatives. Historical records show that Emperor Wu of Liang, Xiao Yan, killed the deposed emperor of Southern Qi, Xiao Baojuan, and took away his palace maid Wu Jinghui, and named her Wu Shuyuan. Wu Shuyuan gave birth to Xiao Zong in July after entering the palace. Therefore, rumors spread that Xiao Zong was actually the posthumous son of Xiao Baojuan. Xiao Yan disagreed, but it became a heartache for Xiao Zong. In the end, Wu Shuyuan told Xiao Zong the truth: Son, your father is Xiao Baojuan. Xiao Zong was very experimental. He dug up his father's grave and dripped his blood on the remains of Xue Baojuan to recognize relatives (dropping bone relatives, blood seeping into the bones is biological, and there is no scientific reason). The blood seeped in. But there was no positive control for this experiment, so Xiao Zong killed his own son who had just turned one month old, took out the bones and conducted further experiments. The blood seeped in, and then he really believed that he was the posthumous son of Xiao Baojuan. This... I think it's a good thing that no one told him that he needed a negative control and repeated experiments.

Homework

How to do the negative control of this experiment? Please leave a message and submit the answer.

Current methods of paternity testing

1. Detection target: microsatellite DNA

The test target that can be used for paternity testing must meet two elements: ① It can be stably transmitted between parents and children, and is stable enough to find their blood relatives; ② It has a high diversity in the population and is unique enough to exclude non-blood relatives. In the human genome, there is a group of DNA fragments, called microsatellite DNA, which can take on this task.

Microsatellite DNA is often called "short tandem repeats" (STR) in animal genetics. It is a type of tandem repeat sequence, which is formed by a short sequence consisting of 1 to 6 bases repeated 5 to 50 times. The human genome contains thousands of STR sequences, and these sequences often exist in non-coding DNA. Compared with other DNA sequences, STR has a higher mutation rate, and the form of mutation is mainly an increase or decrease in the number of short sequence repetitions, which is not eliminated by natural selection (in layman's terms, unimportant things have a lot of room for variation, just like a car wheel can only be round, while the shape of the hub is relatively free). Therefore, STR has a high polymorphism in the population and is called a DNA fingerprint, which is suitable for paternity testing. The identification kit of a mainstream brand uses a combination of 15 STR loci plus 1 sex gene.

The number of STR tests and the selection of loci can be optimized according to different ethnic groups and population sizes, and generally include the thirteen core loci of CODIS. The gender identification gene generally uses the amelogenin gene, which has different versions on the X chromosome and the Y chromosome, called AMELX and AMELY, respectively. AMELY is six bases shorter than AMELX. In this way, if two long DNA products are amplified, it is a female (XX), and if two amplified products, one long and one short, are obtained, it is a male (XY).

Figure: Short tandem repeat sequence STR. The number of short segment repeats at the same STR locus may be different in different people (the figure above shows 7 to 11 repeats). The number and sequence of bases in the short segment repeat units at different STR loci are also different (the base repeat units at the bottom of the figure are 2 to 5). The size of the DNA product fragment produced after primer amplification increases as the product of the number of SRT repeat units and the number of repeats increases (the final amplified fragment includes the sequences on both sides of the STR in addition to the STR sequence, and the total fragment length ranges from 90 to 370 base pairs). [2]

CODIS core STR loci

CODIS (Combined DNA Index System) is a criminal justice DNA database supported by the FBI in the United States. From October 1998 to December 31, 2016, there were 13 core STR loci that needed to be registered during this period. Since January 1, 2017, the number has been expanded to 20.

Figure: The thirteen core STR loci of CODIS and their locations on chromosomes (AMEL is the sex detection gene) [3]

Figure: STR loci selected by a mainstream test kit and their detectable alleles. It includes 13 CODIS core loci and two additional loci, D2S1338 and D19S433, as well as the amelogenin gender identification gene. The value of the allele indicates the number of times the short sequence at the locus is repeated. For example, the D8S1179 locus has 12 alleles, such as 8 and 9. The possible combination of alleles detected by a certain amplification is 12x12=144 (diploid, so each locus has two possible alleles). The possible combinations of each locus can be calculated in turn. The product of all possible combinations of loci is the total number of STR combinations that can be detected by this kit in theory.

Figure: DNA fragment sizes after electrophoresis of all STR alleles that can be detected by a mainstream kit. In actual experiments, all DNA fragments are separated in one lane, and DNA with short fragments runs faster, while DNA with long fragments runs slower, so that DNA fragments of different lengths are separated. Some STR sequence amplification lengths may be the same, in which case they need to be distinguished by the color of the fluorescent dye attached to the fragments.

2. Detection method: PCR

The current STR detection method mainly uses polymerase chain reaction (PCR) to amplify DNA fragments containing specific STR locus sequences and detect their length by electrophoresis to determine the number of STR repeats. The principle of PCR can be found in the second part of the article "Why is it difficult to eliminate false positives in environmental monitoring?"

The current mainstream kit can amplify 15 alleles and 1 sex identification gene in the same test tube. The size of the DNA product fragment produced after amplification with primers increases with the product of the SRT repeat unit and the number of repeats (the final amplified fragment contains not only the STR sequence, but also the sequences on both sides of the STR, and the total fragment length is in the range of 90 to 370 base pairs). The amplified product is separated and detected by capillary electrophoresis.

Figure: Example of test results from a mainstream test kit. One to two peaks can be seen at each STR locus, and the number of allele repetitions can be determined based on the size. If there are two peaks, it means that there are two different alleles at this STR locus, and one of them should be detected in the child or parent. If there is one peak, it means that the two alleles at this STR locus are consistent, and the STR loci of the parents should contain this allele, and their children must have this allele.

3. Test samples: DNA is very stable

Nuclear DNA can be found in any nucleated cell in the body, from saliva to bones, teeth, and hair. A small amount of DNA can be amplified using PCR technology, which can replicate a small number of STR targets in large quantities to achieve the amount of DNA amplification fragments that can be detected. Therefore, as long as the DNA of the subject to be tested can be obtained, no matter what material, it can be used for testing. Usually, the sampling method for paternity testing is similar to the sampling method for the new coronavirus nucleic acid test, except that paternity testing sampling only requires scraping a sufficient amount of autologous cells from the inner wall of the mouth.

DNA is very stable. For some cases where the subject has died, as long as they are not burned to ashes, their hair, bones, and especially the pulp of their teeth can basically be used to obtain DNA samples that can be used for paternity testing. For hair, samples containing hair follicles are far superior to samples without hair follicles, but hair without hair follicles has been reported to be able to be used for paternity testing (mitochondrial DNA or DNA extraction and amplification technology suitable for hair can be used) [4]. If it is impossible to obtain samples from the deceased's body, samples that can be used for testing can also be obtained from the deceased's clothing or personal items (such as combs). In this case, it may be necessary to rule out whether the sample has been contaminated by other people's DNA.

Figure: Chromosomal DNA contained in cells (upper right) and samples that can be used for identification. Starting from the upper left and going counterclockwise, they are hair, saliva, blood, semen, and urine. The last four samples can be obtained from their dried plaques. [5]

The development of modern biotechnology allows non-invasive DNA testing of unborn fetuses. This test is performed by collecting maternal blood through venipuncture to obtain "cell-free fetal DNA" (cffDNA). The so-called cffDNA is fetal DNA that circulates freely in maternal blood and originates from embryonic trophoblast cells. cffDNA analysis is a non-invasive prenatal diagnosis method (screening for genetic diseases, chromosomal abnormalities, etc.) and is often used for older pregnant women. cffDNA that appears after nine weeks of pregnancy can also be used for paternity testing (whether it is allowed by law, you need to consult relevant legal professionals).

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 the first part of "Why is it difficult to eliminate false positives in environmental monitoring?"), but a better electrophoresis analysis platform is required.

5. Analysis of results: may be the final word, or more evidence is needed

After amplification and electrophoresis, the allele type of the detected STR locus can be obtained. The results can be obtained by comparing the allele types of the subjects. Let's take a look at how to analyze through the following simplified example.

The first example is to analyze the DNA test results of the victim, the murder weapon, and the three suspects. The DNA sample on the murder weapon may come from the victim or the criminal. We can see from the band difference that the band on the murder weapon is different from the victim's sample band, which obviously comes from someone other than the victim. Let's compare the murder weapon and the samples of the three suspects. Have you found the murderer suspect?

Figure: Case 1: Who is the murderer? [6]

The second example is a case of identifying the biological father (identifying the biological mother uses the same method and principle). The DNA test bands of the baby in the second column come from the first biological mother and one of the 3rd, 4th, and 5th biological father candidates. For example, the baby's 1st, 4th, and 5th black DNA bands do not exist in the biological mother. This band is inherited from the biological father, while the 2nd, 3rd, and 6th black DNA bands are inherited from the biological mother (the biological mother's bands can match). Next, you just need to find the band that exists in the baby's test results but does not exist in the biological mother, and which candidate father it exists in. Have you found it?

Figure: Case 2: Who is the biological father? [7]

In the actual identification operation, if there is a parent-child relationship between the tested persons, then there is an identical allele at each of their STR loci; conversely, if there is an identical allele at each STR locus of the tested persons, then the possibility of a parent-child relationship is more than 99.99% (the actual operation involves the calculation and application of the Combined Paternity Index, CPI, which is not discussed in this article but does not affect the understanding of the topic).

So, if it is difficult to obtain accurate samples of parents, is it possible to identify siblings through paternity testing?

Regardless of whether they are male or female, as long as their mitochondrial DNA is tested and the sequence is consistent, it can be determined that they are brothers and sisters from the same mother, or cousins ​​from the same maternal line (it is not ruled out that they are distant cousins). If they are brothers, their Y chromosomes are tested and the sequence is consistent, it can be determined that they are brothers from the same father, or cousins ​​from the same paternal line (it is not guaranteed how close the cousins ​​are). As for other chromosomes, or whether the STR loci match, it is within a probability range. For example, if mitochondrial DNA is not considered, theoretically a pair of brothers and sisters can be shown to have no blood relationship in chromosome DNA (the chromosomes selected by sperm and egg gametes are different). In theory, a couple can also give birth to brothers or sisters with completely identical chromosome DNA twice (different ages, but the effect is like identical twins) (the chromosomes selected by sperm and egg gametes are the same). Although the probability of the above situations occurring is very low (considering that chromosomes can also be recombined during the formation of gametes, the situation is more complicated), in real situations, the relationship between brothers and sisters is between the above two extreme situations. Therefore, the credibility of the identification results between brothers and sisters is uncertain, and it is not the best material for blood relationship identification.

Therefore, in real life, paternity testing generally needs to be combined with other evidence (parents' reproductive history, relatives' testimony, or the introduction of more blood-related individuals for identification and analysis) to try to make a more accurate judgment.

6. How was the maternal genetic relationship of the “Chain Woman” determined?

CCTV News screenshot: The report shows that through 60 mtDNA-SNP tests, the people tested are consistent with the maternal genetic relationship. (Source | CCTV News Channel)

On February 23, CCTV News reported the investigation of the "Fengxian woman who gave birth to eight children" incident. The Ministry of Public Security's Forensic Identification Center conducted DNA testing and comparison on the blood samples of "chained women" Yang Mouxia and Guang Mouying and the biological samples extracted from the remains of Xiao Huamei's mother Pu Mouma, and concluded that "Pu Mouma and Yang Mouxia and Guang Mouying are all biologically related parents." The materials shown in the news video show that the object of this test is mitochondrial DNA (mtDNA).

Mitochondria are organelles located in the cytoplasm that perform aerobic respiration and produce ATP, the “energy currency”. Mitochondrial DNA (mtDNA) is DNA located in mitochondria and is an extranuclear genetic material (DNA also exists in plant chloroplasts). Each mitochondria contains multiple copies of circular double-stranded mtDNA. Each mtDNA contains a total of 16,569 base pairs, including 37 genes, which is much smaller than the nuclear genome[8]. For animals, the mtDNA in the fertilized egg is mainly inherited from the mother (the paternal mitochondria will degrade in the fertilized egg). Therefore, the maternal inheritance characteristics of mtDNA allow researchers to trace the long-term maternal genealogy and track maternal ancestors through mitochondrial DNA (the paternal genealogy is carried out using the Y chromosome).

Figure: Mitochondria are organelles located in the cytoplasm of eukaryotic cells, and their matrix contains circular mitochondrial DNA. (Source | Wikipedia)

The mutation rate of mtDNA is higher than that of nuclear DNA, and mutations often occur at the third position of the protein coding sequence codon. In addition, mtDNA has two hypervariable control regions (HVR1 and HVR2), so mtDNA contains a relatively rich number of single-nucleotide polymorphisms (SNPs), that is, the types of bases at a single site are different in the population. Therefore, the number of SNPs contained in mtDNA can be used to analyze the maternal inheritance of the sample being tested. The information of mtDNA was first adopted as evidence by the US court in 1996.

Currently, mtDNA testing can be done by ① measuring HVR1 with the most SNPs, ② measuring the full-length sequence of mtDNA, or ③ using a microarray chip to scan selected SNPs in the entire mtDNA genome. 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 the information from mtDNA alone can only determine the maternal genetic relationship between the tested persons, but cannot determine the relationship between mother and daughter or biological sisters (the so-called maternal genetic relationship between two people means that they have the same great-grandmother in an unknown generation). The two suspected sisters need to obtain the information of their biological mother through separate identification, or as stated in the notice, introduce more test reports with close kinship in order to draw a better conclusion.

CCTV News Screenshot: Expert Opinion (Part) (Source | CCTV News Channel)

In addition, the identification opinion (pictured above) shows that there is a mother-daughter relationship between the suspected biological mother and one of the suspected sisters, Guang Mouying. If the suspected biological mother and the person being tested, Yang Mouxia (the "iron chain girl"), are also related to each other (as explained in the notice), the chain of evidence is complete. The detection method used in this report is nuclear genome STR detection, which detects up to 23 STR loci, which is better than the test kit described in the example in this article.

Conclusion

DNA identification technology has exposed more criminals, corrected more wrongful convictions, reunited more separated families, and revealed more human tragedies. Professionals should regard professionalism as their life, and professional organizations should regard credibility as their life. This world needs the truth, and there must be the truth.

References

[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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