1. Lesson 8 DNA Fingerprinting

2. Insufficient evidence?
Read the following article.
Mr. Chan applied for permission for his son to come to HK to live with him, he had to provide evidence to prove his paternal relationship with his son. He underwent the blood group typing tests and obtained proof that his blood type matched with that of his son. However, Mr. Chan was told by the Immigration Department that the blood group typing result was an insufficient evidence.

3. Insufficient evidence?
Why was the blood group typing result not sufficient evidence?
Any other tests recommend?

4. Blood group typing is much less sensitive
Blood group typing is much less sensitive. May use DNA fingerprint to provide biological evidence as a proof in a paternity test.

5. What is DNA?

6. Structural relationship among chromosomes, DNA and genes
Cells are the basic building blocks of all living things.
In a cell, DNA (deoxyribonucleic acid) is packaged in chromosomes within the nucleus.
DNA is a large, polymeric molecule.
A gene is a segment of DNA molecule of a chromosome. It is the basic unit of heredity. Genes determine the body characteristics of an organism.
Each inherited characteristic is controlled by one or several genes.

7. Look into DNA
DNA looks like an incredibly long twisted ladder. This shape is called a double helix.
The sides of the ladder are a linked chain of 5- carbon sugars and phosphate (PO4) groups (called the backbone).
The rungs connected to the 5-carbon sugars are known as bases.

8. DNA
Each rung is made up of two bases that link together. There are four bases - adenine (A), thymine (T), guanine (G) and cytosine (C).
Because of their chemical nature, A will only link with T and G will only link with C. No base can join with itself (i.e. NO A-A /T- T /G-G / C-C base-pairs).

9. Activity 8.1 What is DNA?
Refer to the Pre-lesson Reading, finish Activity 8.1 to check your understanding on DNA.

10. Activity 8.1
Decide whether the following statements are TRUE or FALSE.
There are 46 pairs of chromosomes in each nucleus of a human cell.
Chromosomes are made of DNA and proteins.
DNA determines the body characteristics of an organism.
DNA may be extracted from red blood cells found in a blood sample.

11. Activity 8.1
The Hair and Teeth of the same person are composed of same DNA molecules.
Identical twins have different base sequences of DNA .
In the DNA structure, adenine (A) will only link with cytosine (C) and guanine (G) will only link with thymine (T).

12. Activity 8.1
Answers:
a. F
b. T
c. T
d. F
e. T
f. F
g. F

13. Activity 8.1
DNA has two strands. And the sequence of one strand determines the sequence of the other. If the base sequence on one strand of DNA is:
GATCCTCATA
What is the base sequence on the other strand?
Answer: GATCCTCATA
CTAGGAGTAT

14. Did you see DNA before?

15. Activity 8.2 DNA Extraction
Objective of experiment
To extract DNA from strawberries with washing-up liquid.
Apparatus and Materials
washing-up liquid (detergent)
salt
ice-cold alcohol
ice bath

16. Activity 8.2 Follow the procedures to extract DNA.
Grind strawberries with a pestle in the mortar.
Add about 10 mL washing-up liquid, 1/2 teaspoon salt and approximately 30 mL tap water to the grinded strawberries. Stir the mixture gently for 1 minute.
Filter the mixture through a muslin cloth into a test tube, about 1/3 full, to separate strawberries from the clear liquid.
Double up the volume of the mixture by adding alcohol slowly into the test tube.
Leave the mixture undisturbed for 2 – 3 minutes in an ice-bath until no emergence of bubbles.
Swirl a toothpick around the interface of the two layers formed inside the test tube. Long and stringy threads can be picked up.

17. Activity 8.2 Questions (optional)
What is the purpose of grinding the strawberries?
To break down the cell wall, cellular membranes and nuclear membranes.
Where is the DNA of the strawberries?
In the cell nucleus.
What is the function of detergent in step 2?
Soap dissolves cell membranes so that DNA is liberated.

18. Activity 8.2 Questions (optional)
What is the function of salt in step 2?
Salt solution helps the DNA strands come together.
What happens when alcohol is added to the solution in step 4?
DNA is soluble in water and will appear clear in water. But DNA is insoluble in non-polar solvent, such as alcohol. Therefore, DNA will precipitate (solidify and appear) out of alcohol. Of course, this is not pure DNA.

19. Follow-up Discussion Write down your answers on a piece of paper.

20. Activity 8.2 Follow-up Discussion Questions
Do you think your results would be different if you use a vegetable or fruit other than strawberries, say, onion, kiwi fruit, broccoli, banana (without skin)? Explain.
Do different organisms have the same DNA?
At a crime scene what evidence would you collect in order to extract DNA? List as many as possible.

21. Activity 8.2
Answers:
DNA can be easily extracted from many different plants. The amount of DNA extracted depends upon many factors, including the number of cells crushed, and whether the cells can be easily broken apart.
Though all DNA molecules are made up of similar chemical components, different organisms have different DNA molecules due to the different base sequences of the bases — A, T, G, and C, which makes a specific organism have distinctive characteristics.
Blood (white blood cells), saliva, skin scalp, hair and etc.
Note that DNA is found in nearly every cell in the body. But one significant exception is red blood cells (lack cell nuclei).

22. Where should you collect DNA evidence?

23. Collection of DNA Evidence
Before the collection of evidence close-ups of any biological evidence should be photographed and its location relative to the entire crime scene needs to be recorded through notes, sketches, and photographs.
All clothing from both the victim and suspect should be collected and sent to the laboratory for examination. Why?

24. Collection of DNA Evidence
Answer:
Because the criminal may have wiped his or her hands on materials not readily apparent to the investigator. In addition, blood may exist in less-than-obvious places.

25. Activity 8.3 Collection & Preservation of DNA Evidence
Guess (i) the possible locations of DNA on the collected evidence from a crime scene and (ii) the possible sources of DNA.
Evidence
Possible locations of DNA on the evidence
Sources of DNA
e.g. Eyeglasses
Ear pieces
Sweat, skin
Baseball bat
Facial tissue
Dirty laundry
Used cigarette
Blanket, pillow, sheet
Bite mark
Fingernail, partial fingernail

26. Possible locations of DNA on the evidence
Activity 8.3
Answers
Evidence
Possible locations of DNA on the evidence
Sources of DNA
Eyeglasses
Ear pieces
Sweat, skin
Baseball bat
Handle
Sweat, skin, blood
Facial tissue
Surface area
Mucus, blood, sweat, semen
Dirty laundry
Blood, sweat, semen
Used cigarette
Cigarette butt
Saliva
Bottle, can, glass
Sides, mouthpiece
Saliva, sweat
Blanket, pillow, sheet
Sweat, hair, semen, urine, saliva
Bite mark
Person’s skin
Fingernail, partial fingernail
Scrapings
Blood, sweat, tissue

27. Preservation of DNA Evidence
Paper bags or other “breathable” containers should be used. Because biologic evidence must never be packaged in airtight containers because moisture can build up, which promotes the growth of bacteria that can degrade DNA.
All precautions against contamination must be taken. These include
wearing face marks, shoe covers and gloves;
using tools such as tweezers to collect evidence.

28. Precautions in handling biological evidence
All body fluids must be assumed to be infectious; hence, wearing disposable latex gloves while handling the evidence is required to prevent being infected.

29. Can DNA be cut?

30. Restriction Enzymes
A common use of restriction enzymes is to generate a "fingerprint" of a particular DNA molecule.
DNA may be cut by some special naturally-occurring proteins called restriction enzymes.
Each restriction enzyme only identifies and “cuts” at a very specific sequence (recognition site) in the DNA strand.
Restriction enzymes typically recognise a symmetrical sequence of DNA, such as the site GAATTC.

31. Example 1
Read to the left
Read to the right 
The top strand is the same as the bottom strand when you read backwards.
When the enzyme EcoRI cuts the strand between G and A, it leaves overhanging chains:
These are termed "sticky ends" because the base pairs formed between the two overhanging portions will glue the two pieces together, even though the backbone is cut.

32. Example 2 GGATCC CCTAGG  G GATCC CCTAG G
Another symmetrical sequence of DNA, GGATCC:
GGATCC
CCTAGG
When the enzyme Bam HI cuts the strand between G and G, it leaves overhanging chains: 
G GATCC
CCTAG G

33. Activity 8.4 Restriction Enzymes
In the worksheet, different DNA samples with their restriction sites are given. Answer the following questions:
How many times does the specified base sequence appear?
How many fragments would result from a restriction digestion of the DNA sample with the given enzyme?
List all the fragments formed.

34. Activity 8.4 Answers: a. The strand is cut into four pieces.
b. Fragments produced:
Hae III,
Bam HI

35. How to separate DNA fragments?

36. Electrophoresis
DNA fragments of different sizes can be separated by using gel electrophoresis.
DNA fragments carry negative charges. When there is a current flow, DNA fragments move towards the anode.
Source: Science Education Section, CDI, EDB.

37. Outline of Electrophoresis

38. Outline of Electrophoresis
Source: Science Education Section, CDI, EDB.
DNA fragments are loaded into “wells” in a gel. The gel floats in a buffer solution within a chamber between two electrodes.
When an electric current is passed through the chamber, negatively charged fragments move towards the positive terminal.
Shorter DNA fragments (smaller size) move faster than the longer ones (bigger size).
In a given time, DNA fragments are separated into bands according to their size.
“wells”
Source: Science Education Section, CDI, EDB.

39. Electrophoresis demonstration
The steps for Electrophoresis are demonstrated in the following video:
(Video Clips: DNA Gel Electrophoresis)

40. DNA Analysis Technique Restriction Fragment Length Polymorphism (RFLP)

41. RFLP It was the first commercial technique of DNA analysis.
It can be used in paternity cases or criminal cases to determine the source of a DNA sample.

42. RFLP Steps It uses restriction enzymes to cut DNA.
DNA fragments of different lengths are produced.

43. RFLP
Using gel electrophoresis, the fragments are separated so that fragments with different lengths can be separated. This provides a pattern of bands.

44. RFLP
Cover specific radioactive probes over the gel. The probes contain a match for the DNA sequence that the test is looking for.

45. RFLP
Put a film under the gel to record. Allow signal exposure in dark room.
DNA fingerprints are now ready for analysis and comparison.
For animation, you may browse
(Virtual Lab.  Topic: 7. RFLP)

46. Another DNA Analysis Technique
To learn more, you may surf the following websites:
Polymerase Chain Reaction (PCR) （
Short Tandem Repeats (STRs) （
Mitochondrial DNA (mDNA) （

47. RFLP Vs PCR
Advantage of using RFLP (more often used in forensic work):
Can individualise a specimen to a narrow portion of the population-possibly one person in billions
Disadvantage of using RFLP:
Requires a larger sample than PCR; more time-consuming and labour intensive; uses radioactive reagents that require special lab. Procedures.

48. PCR Vs RFLP Advantages of using PCR:
Faster and simpler to use and may be applied to exceedingly tiny samples-as small as a billionth of a gram of DNA
Disadvantage of using PCR:
Results of PCR are less dramatic than those of RFLP, being discriminatory on the order of one individual in thousands rather than the potential billions with RFLP
(Crime Science: Methods of Forensic Detection (p.203))

49. What is the use of DNA fingerprinting?
More than 99% of the base sequence in DNA is the same in all humans. Less than 1% is unique to each individual.
Scientists make use of this difference to generate DNA fingerprints which are specific to different individuals.

50. Try to match two DNA fingerprints

51. Warm-up exercise In the following simulation,
decide which two fingerprints match each other;
drag the right-most DNA fingerprint over the suspects’ fingerprints to find a match.

52. Activity 8.5 DNA Fingerprint Analysis
Case 1
Mr. Chan’s family consists of mom, dad and four kids. The parents have one daughter and one son together, another daughter is from the mother’s previous marriage, and the other son is adopted. Here are the DNA analysis results:
Which child is adopted? Why?
Which child is from the mother’s previous marriage? Why?
Who are the own children of Mr and Mrs Chan?

53. Activity 8.5 Answers: Child 4 is adopted.
Child 2 is the child from the mother’s previous marriage.
Child 1 and Child 3 are own children of Mr and Mrs Chan.

54. Activity 8.5
Case 2
A blood sample from a crime scene was collected. DNA samples of the victim and the potential suspects (June, Scarlet and John) were also collected for DNA analysis. The DNA profile is shown. Now, you should be able to identify the potential murderer.

55. Activity 8.5
Answers:
All of the DNA fragments of Scarlet can be found in the crime scene sample making her the most likely suspect.

56. Activity 8.5 Extended discussions:
Both June and Scarlet have the same DNA fragments (“8” and “12”), why?
Why DNA evidence must be combined with the traditional forms of evidence such as eyewitness accounts?

57. Activity 8.5
Answers:
This pattern may arise if the two women are related or if this pattern were common in population.
Someone’s DNA is found at a crime scene does not mean that they committed the crime because of the following reasons:
(i) The DNA sample may be contaminated by the environment.
(ii) The sample may be a mixture of more than one person’s DNA.
(iii) The DNA evidence may be degraded or broken down.

58. Activity 8.6 Ethical and Non-ethical Issues Write down your answers on a piece of paper.

59. Activity 8.6 Group Discussions
Do white blood cells give more accurate test result for paternity than cheek cells?
What are the legal and ethical issues on the use of DNA fingerprinting in society?
DNA fingerprinting can be used to convict someone of a crime. What would be the difficulty in using this technique to identify criminals if the suspects were father and son / twin brothers?
Other than forensic science, list out the other uses of DNA analysis. Describe each use briefly.

60. References
Nickell, J. and Fischer, J.F. (1999). Crime Science: Methods of Forensic Detection. Kentucky: The University Press of Kentucky.