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Polymorphisms

Polymorphisms. Definitions. 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.

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Polymorphisms

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  1. Polymorphisms

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

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

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

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

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

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

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

  9. ..TTATGCCTAACGTACCTGCTAG... ..TTATGCCTAACGTACCTGCTAGCTAACGTACCAGCCCTG... Structural polymorphism: Duplication NOTE: Not all duplications have the exact nucleotidesequence. Two sections are said to be duplicates when90% of the sequence is identical.

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

  11. 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 themand a structural variant.

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

  13. Tools in Molecular Genetics: • Electrophoresis • Probes • Polymerase Chain Reaction • DNA Arrays (Gene Chips)

  14. start lanes - current gel timer + Electrophoresis:

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

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

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

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

  19. PCR: Polymerase Chain Reaction Procedure: • Heat the mixture. Just before the boiling point of water, the DNA will become single-stranded. • 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. • Repeat steps 1 and 2 until a sufficient amount of the desired gene is available for analysis

  20. NewStrands Primers (b) (c) (a) FreeNucleotides

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

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

  23. DNA strand to sequence A T G A C C T C G ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? T A C T G G A G C Primer 1. Heat the DNA to make it single stranded and add a primer. The primer binds to its complementary sequence in the DNA.

  24. C T C T A C A T G A C A C G A G C T DNA strand to sequence G A T C G A T G A G C A T G A C C T C G ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? A T T T A C T G G A G C C T G A C A C T A Primer G T A T G A A G C C T A G T C G C T G T 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.

  25. C T C T A A C T G A C A C G A G C T DNA strand to sequence G A T C G A T G A G C A T G A C C T C G ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? A T T A T A C T G G A G C C T A G A C C T A Primer G T A T A G A A G C C T A G T C G C T T G Polymerase 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.

  26. A T G A C C T C G T A G G C A A A G C C T C G T A T G A C C T C G T A G G C A A A G C C T C G T T A C T G G A G C A T A C T G G A G C A T C C G A T G A C C T C G T A G G C A A A G C C T C G T A T G A C C T C G T A G G C A A A G C C T C G T T A C T G G A G C A T T A C T G G A G C A T C C G T T A T G A C C T C G T A G G C A A A G C C T C G T A T G A C C T C G T A G G C A A A G C C T C G T T A C T G G A G C A T C T A C T G G A G C A T C C G T T T C G A T G A C C T C G T A G G C A A A G C C T C G ? A T G A C C T C G T A G G C A A A G C C T C G T T A C T G G A G C A T C C T A C T G G A G C A T C C G T T T C G G A A A A 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.

  27. T A C T G G A G C A T C C G T T T C G A T G A C C T C G T A G G C A A A G C C T C G ? T A C T G G A G C A T C C T A C T G G A G C A T C C G T T T C T A C T G G A G C A T A C T G G A G C A T C C G T T T C G G A A A A T A C T G G A G C A T C C G T T A C T G G A G C A T T A C T G G A G C A T C T A C T G G A G C A T C C G T T T A C T G G A G C A T C C G T T T C G G A A T G A C C T C G T A G G C A A A G C C T C G ? T A C T G G A G C A T C C G T T T C G G A A T A C T G G A G C A T C C G A T G A C C T C G T A G G C A A A G C C T C G ? T A C T G G A G C A T C C G T T T C G 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.

  28. T A C T G G A G C A T C C G T T T C G G A A A A T A C T G G A G C A T C C G T T T C G G T A C T G G A G C A T C C G T T T C G T A C T G G A G C A T C C G T T T C T A C T G G A G C A T C C G T T T T A C T G G A G C A T C C G T T T A C T G G A G C A T C C G T T A C T G G A G C A T C C G T A C T G G A G C A T C C T A C T G G A G C A T C T A C T G G A G C A T T A C T G G A G C A 6. Use electrophoresis to separate the strands according to size.

  29. 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. T A C T G G A G C A T C C G T T T C G G A A A A T A C T G G A G C A T C C G T T T C G G T A C T G G A G C A T C C G T T T C G T A C T G G A G C A T C C G T T T C T A C T G G A G C A T C C G T T T T A C T G G A G C A T C C G T T T A C T G G A G C A T C C G T T A C T G G A G C A T C C G T A C T G G A G C A T C C T A C T G G A G C A T C T A C T G G A G C A T T A C T G G A G C A

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

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