Polymorphisms

Monomorphism: Section of DNA where the nucleotide sequence is the same for everyone in the population. Polymorphism: Section of DNA with at least two common nucleotide sequences (alleles) in the population. “Common” is arbitrarily defined as a frequency of 1% or more. Definitions Mutant: Allele with a frequency of less than 1%.

Types of Polymorphisms: I. Protein/enzyme polymorphisms ( assay is for the gene product, e.g., blood groups ) II. DNA polymorphisms ( assay the DNA directly ) 1.SNP: Single nucleotide polymorphisms 2.Tandem repeat polymorphisms 3.Structural polymorphisms ( insertions, deletions, inversions, etc. ) CNV = Copy Number Variant 4.Sequencing polymorphisms

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

Tandem Repeat Polymorphisms: A nucleotide sequence is repeated over and over again and the polymorphism is in the number of times it is repeated...TTATGAACGAACGAACGAACGAACGAACGAACGAACTTACGT.....TTATGAACGAACGAACGAACTTACGT... tandem repeat (8 repeat allele) tandem repeat (4 repeat allele) Repeated sequence = GAAC

..TTATGCCTAACTGACTTACCCT.....TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT... Insertion Structural polymorphism: Insertion

..TTATGCCTAACTGACTTACCCT.....TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT... Deletion Structural polymorphism: Deletion

..TTATGCCTAACGTACCTGCTAGCTATACCTGACTTACCCT... Initial Sequence..TTATGCCTAACCCATATCGATCGTCCATGTGACTTACCCT... Inverted Sequence Structural polymorphism: Inversion

..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. Structural polymorphism: Duplication

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

Copy Number Variant (CNV) Long (> 1 kb or > 10 kb) structural variant (insertion, deletion, duplication, inversion, etc). NOTE: Defined by the technology used to detect them, not by any conceptual difference between them and a structural variant.

Redon et al. (2006). Nature 444(23),

Sequencing polymorphisms are the general case SNPs Tandem repeats Structural polys Sequencing polymorphisms

Tools in Molecular Genetics: 1.Electrophoresis 2.Probes 3.Polymerase Chain Reaction 4.DNA Arrays (Gene Chips)

+ gel timer current start lanes - Electrophoresis:

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

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

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

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

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

PCR: Polymerase Chain Reaction

How the Human Genome was Sequenced: (See Text)

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.

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 (dideoxy 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

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

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

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

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

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

ATGCCTGAAATGC CGTTACGTGATGATGCC AATGCGTCATG (a) Unaligned segments: ATGCCTGAAATGC CGTTACGTGATGATGCC AATGCGTCATG (b) Aligned segments: 8. Use computer algorithms to align segments.