DNA Fingerprinting

1. DNA Fingerprinting Class instructions

2. In this lesson your students will learn • DNA fingerprinting uses STR repeats • STRs are repeats of short sequences on DNA • Number of STR copies varies between individuals • Primers must match only one region in the template DNA • Primers must match each end of the target • Complementary base pairing is critical for DNA fingerprinting • How gender is determined from DNA

3. The devil is in the detail! • The 5’ prime and 3’ prime ends of the bases must be round the right way! IMPORTANT: Do not take bases apart!!!

4. Correct base pairing is critical! • Green (Guanine) pairs with yellow (Cytosine) • Blue (Adenine) pairs with orange (Thymine)

5. Using primers to determine PCR product size Primer set 1 Primer set 2

6. Lesson Part One – STR identification • Group 1 and 2 will work out the number of repeats of one STR located on chromosome 16 (see manual for full details). • At this loci people have between 5-16 copies of the STR giving differenet sized products. • The length of the PCR product allows you to count how many copies of the STR repeat are present.

7. Group 1 – STR analysis • Make the double stranded DNA template using bases with black sugars.  Remember to get the 5' to 3' orientation correct! 2 Denature the two strands by separating them. 3 Anneal single stranded primers to the complementary bases - one primer per strand. 4 Work out the length of the final PCR product. How many copies of the STR repeat do you have? • Optional extension – use parts from PCR Puzzle to make a model of the double stranded PCR product using bases with red sugars.

8. DNA Sequence Denature PCR product Anneal primers

9. Group 2 – STR analysis • Make the double stranded DNA template using bases with black sugars: 2 Denature the two strands by separating them. 3 Anneal single stranded primers to the complementary bases - one primer per strand. 4 Work out the length of the final PCR product. How many copies of the STR repeat do you have? 5 Optional extension – use parts from PCR Puzzle to make a model of the double stranded PCR product using bases with red sugars.

10. DNA Sequence Denature PCR product Anneal primers

11. Lesson Part Two - Gender determination • Group 3 and 4 look at gender • X chromosome has a short deletion (see manual for details) • Y chromosome does not have the deletion • Male & female give different size PCR products • You can visualise these on an electrophoresis gel

12. How to determine gender from these results • A female will have XX chromosomes so you get one band on a gel as both give the same size PCR product • A male will have XY chromosomes so you get two bands of different sizes on a gel • Think about how male and female DNA will appear on an electrophoresis gel

13. Group 3 – Gender chromosome 1 Make the double stranded DNA template using bases with black sugars. 2 Denature the template by separating the strands. 3 Anneal single stranded primers to the complementary bases - one primer per strand. 4 Work out length of the final PCR product. 5 Optional extension – use parts from PCR Puzzle to make a model of the double stranded PCR product using bases with red sugars.

14. DNA Sequence Denature PCR product Anneal primers

15. Group 4 – Gender chromosome • Make the double stranded DNA template using bases with black sugars. 2 Denature the template 3 Anneal single stranded primers to the complementary bases - one primer per strand. 4 Work out length of the final PCR product. 5 Optional extension – use parts from PCR Puzzle to make a model of the double stranded PCR product using bases with red sugars.

16. DNA Sequence Denature PCR product Anneal primers