PARENTAGE TESTING

Parentage Testing: testing of genetic markers that are inherited to determine the presence or absence
of a biologic relationship
Parentage is legally presumed that a child born during marriage is also the child of the husband. The
presumption can be rebutted. The court can order DNA or other tests, but if the mother, having control,
does not allow samples to be taken then all the court can do is draw such inferences as are appropriate.
There are very technical rules as to who is a parent in cases of artificial insemination and the like.

Purposes:
1. to answer the question whether a man is the biologic father of a child
2. to determine maternity or siblingship

History
- 1924  The three allele theory
- 1926  ABO blood group testing
- 1955 ( Smithies) described the polymorphism of haptoglobin, opening the door to a new type of
genetic system, the enzymes and proteins
- 1972 : ( Evan Workshop organized by Dausset)
-
All of the genetic systems were expressed on different elements of the blood
1. Red Blood cells
2. Proteins
3. Leukocytes
One in common : They were all dependent on a complex biochemical expression of the original genes.
The group of genetic system is often termed the “ Classic System”
- 1985  New type of polymorphism in human DNA hyper variable mini-satellites often referred
as VNTR ( Variable number tandem repeats)
 VNTR systems were tested by treatment of the extracted DNA with restriction enzymes
known as RFLF Restriction Fragment Length Polymorphism
 Followed by: Southern Blotting
 Then PCR ( Polymerase Chain Reaction)

Application of VNTRs

 VNTRs are an important source of RFLP genetic markers used in linkage analysis (mapping) of
genomes. They have become essential in forensic crime investigations. The technique may use
PCR, size determined by gel electrophoresis, and Southern blotting to produce a pattern of
bands unique to each individual.
 Therefore, VNTRs are being used to study genetic diversity (DNA fingerprinting) and breeding
patterns in animals. VNTRs also have clinical applications like VNTR typing, the next gold
standard in genotyping for early diagnosis of M. tuberculosis super-infection or mixed infection.
VARIABLE NUMBER TANDEM REPEATS (VNTRS)

 VNTR are structural regions of the DNA where a short sequence of nucleotidesare structural
regions of the DNA where a short sequence of nucleotides (longer than 3) is repeated a variable
number of times in tandem.
 VNTRs are commonly subdivided into microsatellites (repeat sequences shorter than 5
nucleotides) and minisatellites (repeat sequences larger than 5 nucleotides) and, like triplet
repeats, are thought to be due to DNA slippage errors during DNA replication.
 As the number of repeats is very individually determined (and generally differs between the
maternal and paternal copy), analysis of VNTRs is often used in forensic and paternal identity
research.

POLYMERASE CHAIN REACTION (PCR)

 Developed in 1983 by Kary Mullis

 Awarded Nobel Prize in 1993
 PCR is shorthand for a simple but very useful procedure in molecular biology called the
polymerase chain reaction.
 It is a technique used to amplify a segment of DNA of interest or produce lots and lots of copies.
In other words, PCR enables to produce millions of copies of a specific DNA sequence from an
initially small sample – sometimes even a single copy. It is a crucial process for a range of
genetic technologies and, in fact, has enabled the development of a suite of new technologies.

How does PCR works?

 PCR mimics what happens in cells when DNA is copied (replicated) prior to cell division, but it
is carried out in controlled conditions in a laboratory. The machine that is used is simply called
a PCR machine or a thermocycler. Test tubes containing the DNA mixture of interest are put into
the machine, and the machine changes. the temperature to suit each step of the process.
 Standard ingredients in the mixture are:
a. the DNA segment of interest
b. specific primers
c. heat-resistant DNA polymerase enzyme
d. the four different types of DNA nucleotides
e. the salts needed to create a suitable environment for the enzyme to act.

PCR Process
Step 1: Denaturation
 As in DNA replication, the two strands in the DNA double helix need to be separated.
 The separation happens by raising the temperature of the mixture, causing the hydrogen
bonds between the complementary DNA strands to break. This process is called
denaturation.
Step 2: Annealing
 Primers bind to the target DNA sequences and initiate polymerisation. This can only occur
once the temperature of the solution has been lowered. One primer binds to each strand.
Step 3: Extension
 New strands of DNA are made using the original strands as templates. A DNA polymerase
enzyme joins free DNA nucleotides together. This enzyme is often Taq polymerase, an
enzyme originally isolated from a thermophilic bacteria called Thermus aquaticus.
 The order in which the free nucleotides are added is determined by the sequence of
nucleotides in the original (template) DNA strand.

 The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing
one newly made strand and one original strand.
 The cycle is repeated many times (usually 20–30) as most processes using PCR need large
quantities of DNA. It only takes 2–3 hours to get a billion or so copies.
 PCR technology is still developing. There is continuing development and refinement of the
processes and tools used, allowing the process to be adapted to meet specialist needs. For
instance, new methods and refinements are being developed and used, especially when
quantification of DNA in a sample is needed.
 New methods include real-time PCR or quantitative PCR (qPCR) and digital PCR (dPCR). In
qPCR, the amplification of DNA is monitored in real time, allowing the quantification of target
DNA throughout the process. dPCR is a new, more refined approach that breaks the PCR
process up into many smaller steps. It offers increased precision, more reliable measurements
and absolute quantification from very small or mixed samples.

PCR Basics

 Isolate and mass produce a particular DNA fragment

a. Template DNA
b. Polymerase – enzyme that replicates DNA, matches complementary bases
c. Primers – 10-30 base pairs long, complementary to ends of fragment

a. Denature
b. Anneal
c. Extend
SHORT TANDEM REPEATS
- Short regions of DNA that differ substantially
among people
 TCAT
- Each person carries a unique combination of
repeats

Applications
- Implicating a crime suspect, acquitting the
wrongly convicted
- Paternity
- Identifying the deceased

Obtaining DNA
- Any biological material
 Body tissues
 Body fluids
 Hair follicles
 Dried material

POWER OF DISCRIMINATION

 Ability to discriminate between different individuals

 The larger the number of loci used, the more powerful the ability to discriminate

Paternity Testing (Exclusion of Parentage)

1. Sociological Aspects
a. Born out of wedlock
b. Disputed Parentage
c. Newborns kidnapped from hospital nurseries
d. Missing Children
e. Children abducted by non-custodial parents/strangers

 Reason: to define the status of an illegitimate child

1) Child Support
2) Inheritance
3) Custody
4) Birth Records
5) Welfare Laws ( a non marital child may make claims and be compensated as a
dependent)

 Blood Group System is a set of MARKERS encoded by allelic genes that occupy a locus either
of a pair of homologous chromosomes
 Genetic Markers: recognizable characteristics inherited from parents and controlled by alleles
on a pair of chromosomes.
 - A pair of homologous chromosomes (one from mom, one from dad); Humans have 23 pairs of
homologous chromosomes
- Gene : unit of DNA information about a trait passed from parents to offspring specific location
on a chromosome
- Alleles – different versions of a gene
- Chromosomes are structures located within the cell nucleus that carry genes in a linear order
as part of the DNA molecule.
 Mother (Ovum) Father (Sperm cell)  Gamete (carries 1 chromosome of each pair)

2. Mendel’s Basic Rules of Inheritance

 Unit Inheritance: Transmitted through generations intact
 Allelic Segregation: A pair of genes ( one obtained from each parent and found on a separate
chromosome) is never found in the same gamete but is always separate.
 Independent Assortment: Different pairs of genes assort independently of each other unless
the different genes are closely linked on the same chromosomes.

Criteria for Selection of Genetic Systems

1. The system should be polymorphic: that is, it should have multiple alleles, ideally in Hardy-
Weinberg equilibrium
2. Inheritance pattern should be established and follow Mendel’s Rules
3. Mutation rate should be known to be low
4. Possible genotypes should be easily deducible from the phenotype
5. Testing methodology should be reliable, reproducible and available in more than one laboratory
6. Markers tested should be stable and not affected by environmental factors, age, disease,
reagents, or methodology employed.
7. Databases of allele frequencies should be available for all ethnic groups that may be tested.
8. Allelic distributions should be able to provide a high probability of excluding a falsely accused
man
9. Each genetic system should be known to be genetically independent ( e.g., no linkage
disequilibrium) of all other systems selected by the laboratory.

3. General Principles
1. Each person inherits one paternal and one maternal allele for blood factors
2. In logical interpretation of inheritance, a person can be either homozygous or heterozygous for
each blood factor.
3. In some blood factors, it is possible to distinguish between homozygous and heterozygous,
whereas for other blood factors, this is not possible.
4. A person can’t possess a blood factor that is absent from the father and from the mother.
5. A blood factor cannot be absent in a person if one of his parents is homozygous for the factor.
6. If a parent is heterozygous for a factor of which both allele can be demonstrated by suitable
test, his child must possess a blood factor corresponding to one of these two alleles.

4. Laws
1. ABO
 Blood factors A or B cannot appear in a child unless present in one or both parents.
 Parents of Group” AB” cannot have a child of Group “O”.
 Parents of Group “O” cannot have a child of Group”AB”
 2. MN
 A child cannot possess M and N unless these factors are present in the blood of one or both
parents.
 A parent of Type “M” canot have a child of type “N”.
 A parent of Type “N” cannot have a child of Type “M”.

3. Rh Considerations
 None of the Rh factors (Rho or (D), rh’(C ) rh”(E), hr’(c ), hr”(e) can be present in a person
unless one or both parents possess corresponding factors.
 A parent lacking the rh’(C ) factor cannot have a child without hr’(c ) factor or vice versa.

5. Factors considered
 Proper Performance
 Sensitiveness of the test (Potency, Reliability)
 Expertness of the Medical Technologist

6.0 Validity of the tests must be secured by the following manner:

 Tests- duplicate
 Each blood factor must be tested with at least two different kinds of antiserum and concordant
results must be obtained.
 All antiserum used must be tested with control specimens of known red cells
1) with corresponding antigen
2) without the corresponding antigen.

7. PPE (Prior Probability of Exclusion)

 The greater the power of the system, the less likely that a falsely accused man will remain
unexcused by the tests.
1) Listing of phenotypes of all possible trios ( Mother, Child and possible father) that will
result in an exclusion of the alleged father.
2) Using genotype/phenotype frequency charts for a given racial group.
3) Combining the results for all possible trios in a system.
4) Combining the results for all systems used.

8. PPE Systems
a. Blood Group Systems
1) ABO
2) MNSs
3) Rh-Hr
4) Duffy
5) Kidd
6) Kell
b. RBC enzymes/Serum Proteins
c. Immunoglobulin Allotypes
1) Gm Polymorphism – gamma heavy chain
2) Km- the kappa light chain
3) Am- the alpha heavy chain
 d. The HLA system
 The HLA complex represents the most polymorphic genetic system in the human genome
 Class I
 Class II antigens
 Only antigens expressed by the A and B loci are considered
 The usual method is “ microlymphotoxicity”. It depends on the evaluation of the reactions of
live lymphocytes with a panel of cytotoxic antibodies of known specificity in the presence of
complement.
1) Testing is done: 60 to 722 microwells trays preloaded with reagent antisera
2) A sufficient number of antisera should be used so that HLA-A and HLA B so
specificities can be identified
3) Additional antigens should be tested if appropriate antisera can be obtained. The
larger the number of antigens that can be defined, the more powerful the system
becomes, that is, the better it is able to exclude falsely accused men.
Source of antisera
1) Human
2) Monoclonal ( Monospecific antisera are not available for a large number of antigens)

Requirement

 Each antigen be defined by at least two different operationally monospecific ( by one

monospecific sera and two multispecific sera or by three multispecific sera.
Terminology

 When an inconsistency in the inheritance pattern is detected in a genetic system result

concerning a DNA polymorphism, the term “ exclusion” is not used.
 The term “ mismatch” is used to refer to the fact that the bands between the child and the alleged
father do not match.
 The term “ exclusion” is reserved for the final interpretation of the entire set of genetic systems
tested.
 A minimum of two mismatches is required before an opinion of non-paternity ( non maternity) is
rendered.

DIRECT EXCLUSION: occurs when a marker is detected in the child but is absent in the mother and the
alleged father.
Examples:
1) The child has blood group B, the mother has group A and the alleged father is group O.
2) The alleged father has “AB” and the Child is “O”

INDIRECT EXCLUSION: occurs when a single marker is detected in the child and a different single
marker is detected in the alleged father. Based on the assumption that if a person expresses only one
marker.
Paternity testing

 Although blood group studies cannot be used to prove paternity, they can provide unequivocal
evidence that a male is not the father of a particular child. Since the red cell antigens are
inherited as dominant traits, a child cannot have a blood group antigen that is not present in one
or both parents. For example, if the child in question belongs to group A and both the mother
and the alleged father are group O, the man is excluded from paternity.
 The table shows the phenotypes (observed characters) of the offspring that can and cannot be
produced in the matings on the ABO system, considering only the three alleles (alternative
genes) A, B, and O. Similar inheritance patterns are seen in all blood group systems.
 Furthermore, if one parent is genetically homozygous for a particular antigen—that is, has
inherited the gene for it from both the grandfather and grandmother of the child—then that
antigen must appear in the blood of the child.
 For example, on the MN system, a father whose phenotype is M and whose genotype is MM (in
other words, a man who is of blood type M and has inherited the characteristic from both
parents) will transmit an M allele to all his progeny.
Analysis
 The child is (-) for two different allelic antigens both of which are present in the alleged father
 Ex.
 Alleged Father “AB” Genotype A and B
 Child “O” Genotype O/O
 - A and B genotype indicated the A antigen from 1 parent and the B antigen from the other.
- One antigen and or the other must be present in the child
- The child appears homozygous for an antigen demonstrated in the mother but not present in the
alleged father.
 Alleged F “N” Genotype N/N
 Mother “M” Genotype M/M
 Child “M” Genotype M/M

Analysis: The alleged father appears homozygous for an antigen not present in the child.
 Alleged F “CDe” Genotype CDe/CDe
 Mother “cde” Genotype cde/cde
 Child “cde” Genotype cde/cde

Mother “O” Genotype O/O

Alleged F “A1” A1O, A,A,, A,A2
 Child 1 “O” OO
 Child 2 “A1” A,O
 Child 3 “A2” A2O

Analysis:
 If father is A,O, A,A, his children arechild 1 and 2
 If father is A1A2, his children are child 2 and 3.