1. Types of Polymorphisms I. Protein/enzyme polymorphisms Blood groups II. DNA Polymorphisms 1.Single Nucleotide Polymorphisms (SNP) 2.Tandem Repeat Polymorphisms 3.Structural Variants Insertion/Deletion/Inversion/Duplication/Translocation Copy Number Variants (CNV) Microsattelites, Short Sequence Repeats (SSR) Variable number of tandem repeats (VNTR)

2. SNP: Single Nucleotide Polymorphism Section of DNA that difference in one and only one nucleotide. TGATCTTG...........TGCCAGTT......... CCGTAGCGAA TGATCTTG...........TGCTAGTT......... CCGTAGCGAA Allele 1: C Allele 2: T

3. Tandem Repeat Polymorphisms: A nucleotide sequence is repeated over and over again and the polymorphism is in the number of times it is repeated.

4. ..TTATGAACGAACGAACGAACGAACGAACGAACGAACTTACGT.....TTATGAACGAACGAACGAACTTACGT... tandem repeat (8 repeat allele) tandem repeat (4 repeat allele) Repeated sequence = GAAC

5. ..TTATGCCTAACTGACTTACCCT.....TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT... Insertion Insertion Polymorphism

6. ..TTATGCCTAACTGACTTACCCT.....TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT... Deletion Deletion Polymorphism

7. Inversion Polymorphism..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT... Initial Sequence..TTATGCCTAACCCATATCGATCGTCCATGTGACTTACCCT... Inverted Sequence

8. Duplication Polymorphism..TTATGCCTAACGTACCTGCTAGCTAACGTACCAGCCCTG.....TTATGCCTAACGTACCTGCTAG... NOTE: Not all duplications have the exact nucleotide sequence. Two sections are said to be duplicates when 90% of the sequence is identical.

9. Translocation Polymorphism (A) Section of a chromosome breaks off CTGACTTACCCT.....AGTCGCTAGATCTA..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT... CTGACTTACCCT.....TTATGCCTAACGTACCTGCTAGCTATAC (B) Broken segment attaches to another chromosome (often at a telomere)

10. Copy Number Variant (CNV) (1)Somewhat long (> 1kb) section of DNA that is repeated throughout the genome with variable copy numbers. (2)Repeats do not have to be in tandem. (3)Includes long insertions, deletions, and duplications.

11. Redon et al. (2006). Nature 444(23), 444-454.

12. Tools in Molecular Genetics: 1.Electrophoresis 2.Probes 3.Polymerase Chain Reaction 4.Restriction Enzyme 5.Dideoxy Nucleotides 6.DNA Arrays (Gene Chips)

13. + gel timer current start lanes - Electrophoresis:

14. http://www.ucl.ac.uk/~ucbhjow/b241/biochemical.html

15. GAATTC...GACTTC...GAATTC... TTAAG...C CTTAAG... Probe: Section of single-stranded DNA (or RNA) that binds to complementary DNA and carries a “lightbulb”

16. PCR: Polymerase Chain Reaction Purpose = Make a lot of copies of a desired piece of DNA (i.e., “amplify” the DNA)

17. PCR: Polymerase Chain Reaction Start with a soup containing: (1)the DNA that you want to amplify (2)enzymes to replicate DNA (polymerase) (3)lottsa free nucleotides (4)primers = short initial section of the gene that you want to amplify (e.g., ) C A A A C C CC G T T T T T G GG G G GATCCAG C C T T A A G

18. PCR: Polymerase Chain Reaction Procedure: 1.Heat the mixture. Just before the boiling point of water, the DNA will become single-stranded. 2.Cool the mixture. As the mixture cools, the primer will bind to the DNA and the polymerase will synthesize a new strand for each strand of DNA. 3.Repeat steps 1 and 2 until a sufficient amount of the desired gene is available for analysis

19. (a) Primers (b) New Strands Free Nucleotides (c)

20. http://www.britannica.com/nobel/cap/opolchr001a4.html PCR: Polymerase Chain Reaction

21. RFLP: Restriction Fragment Length Polymorphism Polymorphism based on whether or not a restriction enzyme cuts a section of DNA. Usually two alleles: (1) the restriction enzyme does cut in the middle of the genes; and (2) the restriction enzyme does not cut in the middle of the gene. restriction enzyme = enzyme that recognizes a specific nucleotide sequence and cuts the DNA at that sequence.

22. GAATTC...GACTTC...GAATTC... AATTC...GACTTC...G Allele 1: restriction enzymes GAATTC... Allele 2: AATTC...G G

23. GACTTC...G TTAAG... Probe: C CTTAAG... AATTC...G TTAAG... Probe: C CTTAAG... AATTC...G TTAAG... Probe: C CTTAAG... (c)

24. Starting Lanes Moe LarryCurly Homozygote: neither allele cut, so two long strands Heterozygote: One long strand from the allele that was not cut, and two strands from the allele that was cut Homozygote: two alleles which the restriction enzyme cut

25. The “Olden” Days

26. Current Technology 1.PCR that includes dideoxy nucleotides with ordinary nucleotides. 2.Have a laser scan the fragments from electrophoresis. dideoxy nucleotides = “color coded” nucleotides (e.g., A, T, C, G ) that stop the synthesis of a new DNA chain when they are inserted into the chain.

27. http://www.sanger.ac.uk/genetics/exon/ Finding a SNP

28. http://www.wtcrf.ed.ac.uk/genetics/images/snp1.jpg SNP Genotyping Machine (7000 genotypes per day) Wellcome Trust Clinical Research Facilities

29. Potential New Method: Nanopores

31. How the Human Genome was Sequenced: (See Text)

32. TACTGGAGC ATGACCTCG??????????????? DNA strand to sequence Primer 1. Heat the DNA to make it single stranded and add a primer. The primer binds to its complementary sequence in the DNA.

33. 2. Add nucleotide alphabet soup. Two types of nucleotides are in the soup. The first (black letters) are ordinary nucleotides. The second (colored letters) are special nucleotides that have two important properties: (1) they will halt the synthesis of the DNA strand whenever they are incorporated into it, and (2) they will fluoresce when viewed under the appropriate lighting. TACTGGAGC ATGACCTCG??????????????? DNA strand to sequence Primer A A A A A A A A A A A A A A A A TT T T T T T T T T T T T T T T C C C C CC C C C C C C C C C G G G G G G G G G G G G G G

34. 3. Add the polymerase (an enzyme that adds free nucleotides to the primer strand). The polymerase will “grab” free nucleotides and add the appropriate one to the extend the strand. TACTGGAGC ATGACCTCG??????????????? DNA strand to sequence Primer A A A A A A A A A A A A A A A A TT T T T T T T T T T T T T T T C C C C CC C C C C C C C C C G G G G G G G G G G G G G G A A Polymerase

35. 4. Complementary strands will be synthesized, but they will be of different lengths depending on where the colored nucleotide is incorporated. Eight examples are given below. TA ATACTGGAGC ATGACCTCGGGCAAAGCCTCGT TA ATACTGGAGC ATGACCTCGGGCAAAGCCTCGT T TA ATACTGGAGC ATGACCTCGGGCAAAGCCTCGT TC TA ATACTGGAGC ATGACCTCGGGCAAAGCCTCG? TCC TA ATACTGGAGC ATGACCTCGGGCAAAGCCTCGT TCCG TA ATACTGGAGC ATGACCTCGGGCAAAGCCTCGT TCCGTT TA ATACTGGAGC ATGACCTCGGGCAAAGCCTCGT TCCTTTCGG TA ATACTGGAGC ATGACCTCGGGCAAAGCCTCGT TCCTTTCGGGAAAA

36. 5.Heat the DNA to make it single-stranded. There will be many copies of the template strand and also many copies of different length of the synthesized strands. ATACTGGAGC ?TAATGACCTCGGGCAAAGCCTCG TAACTGGAGCTTAACTGGAGCTC ATACTGGAGCTCC ATACTGGAGCTCCG ATACTGGAGCTCCGT ATACTGGAGCTCCTTTCGG ATACTGGAGCTCCTTTCGGGA ATACTGGAGCTCCTTTCGGGAAAA ?TAATGACCTCGGGCAAAGCCTCG ATACTGGAGCTCCTTTCGGGAA ATACTGGAGCTCCTTTCG ATACTGGAGCTCCTTG ATACTGGAGCTCCTTTCGG ?TAATGACCTCGGGCAAAGCCTCG

37. 6. Use electrophoresis to separate the strands according to size. ATACTGGAGC TAACTGGAGCT TAACTGGAGCTC ATACTGGAGCTCC ATACTGGAGCTCCG ATACTGGAGCTCCGT ATACTGGAGCTCCTTTCGG ATACTGGAGCTCCTTTCGGG ATACTGGAGCTCCTTTCGGGAAAA GCATACTGGACTCTTTG ATACTGGAGCTCCTTG ATACTGGAGCTCCTTTCG

38. 7. Viewing the gel under a special light allows the colored nucleotides to fluoresce. This lights up the band. The color-coding permits the DNA sequence to be read. ATACTGGAGC TAACTGGAGCT TAACTGGAGCTC ATACTGGAGCTCC ATACTGGAGCTCCG ATACTGGAGCTCCGT ATACTGGAGCTCCTTTCGG ATACTGGAGCTCCTTTCGGG ATACTGGAGCTCCTTTCGGGAAAA GCATACTGGACTCTTTG ATACTGGAGCTCCTTG ATACTGGAGCTCCTTTCG

39. ATGCCTGAAATGC CGTTACGTGATGATGCC AATGCGTCATG (a) ATGCCTGAAATGC CGTTACGTGATGATGCC AATGCGTCATG (b)