MDSC/DENTC1104: Introduction Molecular Medicine

DNA/ Paternity Lab

Due Tuesday October 18 at 1pm.
Collection Venue: Outside of Lecture Theatre 3
PART A

1. What are the steps involved in processing DNA samples to determine paternity?
NB. Please provide detailed explanation of each step (35 marks)

STEPS INVOLVED IN PROCESSING DNA SAMPLES FOR PATERNITY

Introduction:
DNA is the genetic material that needs to be tested. It can be tested for several things. It can

be used to identify genetic mutations. DNA may also be used to assist in criminal

investigations. Additionally, it is used to establish a value for a child's paternity. Paternity

tests can be very accurate almost 100%. It is done using something known as an STR test.

STR stands for short tandem repeats. These repeats found on the gene are the basis used for

paternity testing.

Step 1: Collection of the sample

This could be done in two ways, blood tests or a less invasive cheek swab. The

father and child will be in a medical facility and must consent to take the blood test or

swab. The blood may be taken from the arms of the family. The swab is taken from

the cheeks of the patients by the patients or a licensed practitioner. One swab must be

done for each cheek and each should last approximately 30 seconds.

Step 2: Extracting the DNA

 The extraction of the DNA will differ based on the sample, whether blood or

tissue. The cells must be lysed to obtain their internal contents. Lyse comes from the

word lysis which means to disintegrate. Lysing is a process by which the cell is

broken open and the DNA within is retrieved. It may be done thermally, chemically,

or even with the use of enzymes for example Proteinase K.

PCR amplification of multiple STR loci simultaneously, or multiplexing utilizes

different coloured flurescent dyes

Step 3: Separating DNA

The DNA obtained may be contaminated or mixed around with debris and

chemicals that may have been used in the previous step. It must be isolated and

purified. DNA is then precipitated using sodium ions which will make the molecules

less water soluble and help with stability. Alcohol is added and causes the DNA to

precipitate out of the aqueous solution because it isn’t soluble in alcohol. Adding a

protease will degrade any DNA-associated proteins. Purification is the next step. The

DNA is rinsed with alcohol, specifically isopropanol or absolute ethanol, removing

the rest of the unwanted debris. Then it is dissolved in water for proper storage. The

most popular method of separation though is electrophoresis because DNA is

negatively charged. This uses an electric field to move molecules through a gel-like

substance or matrix. They migrate to the positive electrode and as smaller fragments

move faster; the larger ones move slower. They are arranged in order of size and the

bands formed are compared throughout the family.

Step 4: Genotyping using PCR

 After separation, the DNA is copied using the PCR technique.

PCR is a process that produces millions of copies of a selected DNA sequence. DNA

polymerases and 2 primers are needed as well as large amounts of the 4 DNA

nucleotides. The primers are needed as a starting point for the new strand so the DNA

polymerase can go to work. The DNA that has been stored is then heated to separate

the double strand into single strands. It is cooled and the primers begin to bind to their

complement sequences. This is done to give a small segment of the DNA that is

desired. It’s then heated once more as the polymerases build new strands of the DNA

by attaching the nucleotides until the segment is filled. Now you have the specific

copied sequence needed for the study.

Step 5: Testing

These copies are Tested for genetic markers. Loci which are the locations of

these markers house two alleles that are from each parent. A comparison must be

made to the population database based on the phenotype the individual displays. After

the known genetic markers are located, they are compared and matched between the

child and parent/s. A paternity report is prepared.

2. What is a paternity report and how is it analyzed? (10 marks)

A paternity report is a DNA test utilized to determine the relationship between a

mother, an alleged father and their child but is not limited to this portion of the family.

On your paternity test you’ll have the index, the probability of paternity and test

conclusion the table contains all the data results in the report. At least 15 different

DNA locations are listed in the locus column and then compares the data with the

same locations for other tested parties. A child inherits one copy of the DNA segment
 from the mother and one from the father known as alleles. the number associated with

each allele represents the length of the DNA segment where the man tested is

considered the biological father of the child tested. the possible father and the child

must match one allele at each genetic location, except in cases where a genetic

mutation exists. when there are mismatches at most locations then the result will be

called an exclusion meaning the man tested is not considered as the biological father.

If only one allele is present at a single location, it means the two alleles from each

parent is the same length.

Pi stands for paternity index. the Pi column on the table depicts the strength of the

genetic match between alleged father and child at that location. the higher the Pi the

stronger the match. multiply each Pi is multiplied together to obtain the combined

paternity index or CPI. this ratio indicates how many times more likely it is that the

man tested is the biological father than someone randomly selected from the

population with the same ethnicity. the higher the CPI the stronger the results. if a

man is excluded as biological father then the CPI is always 0. the probability of

paternity is based on the combined paternity index and is calculated using

mathematical formula. if the man tested is not excluded as the biological father then

the percentage of probability is 99.9% or higher if he is excluded as the biological

father, the probability of paternity is 0%. The probability can never be 100% because

each man from the child’s phenotypical race would have to be tested as well.

PART B

3. Please complete the table below with the data from cases A & B.

CASE 1: PATERNITY TABLE FOR ALLEGED FATHER AND CHILD ONLY

 Locus Child Alleged Shared Frequency Formula Paternity
Father Allele of Allele Index (PI)
CSF1PO 7,10 7 7 0.04286 1/2q 11.666
D5S818 8,12 8,13 8 0.05 1/4q 5.000
D7S820 9,10 9,10 9,10 0.15714, (p+q)/4pq 2.363
0.32381
TPOX 11 11 11 0.22488 1/q 4.447
D18S51 16,19 16,21 16 0.18889 1/4q 1.324

CASE 2: PATERNITY TABLE FOR ALLEGED FATHER AND CHILD ONLY

Locus Child Alleged Shared Frequency Formula Paternity
Father Allele of Allele Index
D21S11 31, 33.2 31, 35 31 0.09218 1/4q 2.712
D5S818 13 15 None 1 0 0.000
THO1 6, 8 6, 9 6 0.10952 1/4q 2.283
Vwa 16 18,20 None 1 0 0.000
D18S51 13,18 16,20 None 1 0 0.000

(40 marks)

4. Determine the PI, combined PI and the probability of paternity for cases A&B.
(30 marks)
Combined PI= Product of all the PIs of the loci used

CPI for A =11.666 x 5.000 x 2.363 x 4.447 x 1.324

CPI for A = 811.542

Probability of paternity:
(CPI x 0.5)/ [(CPI x 0.5) +0.5] x 100
811.542 x 0.5)/ [(811.542 x 0.5) + 0.5] x 100
= 99.88%

CPI for B = 2.712 x 0.000 x 2.283 x 0.000 x 0.000

CPI for B= 0

Probability of paternity
0%

References (APA format) (5 marks)

Endeavor DNA. (2021, September 1). What is the process for DNA testing? Endeavor DNA.
Retrieved October 16, 2022, from https://endeavordna.com/blog/process-dna-test/

Total marks: 120