1 / 22

The Human Genome Project

The Human Genome Project. And how we got there… Sequencing technologies Sequencing strategies So what? What’s next. But before that…. How do you find out the sequence of DNA? Sanger’s dideoxy sequencing method. Frederick Sanger. Won the Nobel Prize in Chemistry twice

macy
Télécharger la présentation

The Human Genome Project

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. The Human Genome Project And how we got there… Sequencing technologies Sequencing strategies So what? What’s next

  2. But before that… • How do you find out the sequence of DNA? • Sanger’s dideoxy sequencing method

  3. Frederick Sanger • Won the Nobel Prize in Chemistry twice • 1958 – for sequencing insulin • 1980 – for inventing a method for sequencing DNA (together with Gilbert) • All the high-throughput sequencing methods in use today are based on the Sanger dideoxy method http://www.nlm.nih.gov/visibleproofs/media/detailed/vi_a_208b.jpg

  4. Sanger’s recipe: Ingredients • The DNA of interest - template • An oligonucleotide primer to get the ball rolling • A DNA polymerase • dNTPs (deoxyribonucleotide triphosphates) – dATP, dCTP, dGTP, dTTP • The special ingredient: ddNTPs

  5. Revision from ATBMS: Nucleosides Triphosphates/deoxyribonucleotide triphosphates Concepts of Genetics 7th Ed, Klug and Cummings

  6. Revision from ATBMS: Nucleosides Triphosphates/deoxyribonucleotide triphosphates • Phosphodiester bonds are formed between the 3’ carbon of one nucleotide and the 5’ carbon of the next nucleotide Concepts of Genetics 7th Ed, Klug and Cummings

  7. Linkage of two nucleotides Revision from ATBMS: Nucleosides Triphosphates/deoxyribonucleotide triphosphates Concepts of Genetics 7th Ed, Klug and Cummings

  8. What’s special about ddNTP? This method is also known as the chain termination method Concepts of Genetics 7th Ed, Klug and Cummings

  9. What’s special about ddNTP? Fluorescent dye coupled to N-base Each ddNTP - ddATP, ddCTP, ddGTP, ddTTP– is coupled to a different type of fluorescent dye – each ddNTP will absorb a characteristic laser wavelength and emit a characteristic colour

  10. Sanger recipe: Method • Divide DNA into 4 tubes with dNTPs and a different ddNTP in each tube and incubate • Polymerase catalyses addition of dNTPs • ddNTPs will terminate reactions • Form oligonucleotides of varying lengths terminated by fluorescent ddNTPs

  11. Denature DNA to produce single stranded oligonucleotides Load single stranded oligonucleotides and separate by electrophoresis – usually by capillary electrophoresis ‘Read’ DNA sequence What would an agarose gel look like?

  12. Advances in technology… • The use of fluorescently labelled ddNTPs (previously radioactive isotopes were used) • Each ddNTP could be labelled with a different flurochrome • Sequencing could be done in a single tube • Capillaries replaced large sheet gels • Fluorescence could be read by a laser, leading to: • Automation • The human genome was sequenced using Sanger’s dideoxy method

  13. Capillary electrophoresis (from wikipedia) Capillary tube filled with agarose and buffer Electrical voltage applied across the capillary Oligonucleotides move across capillary, according to size

  14. Typical Electropherogram But usually first 10-20 bp are not reliable, also limited to about 600-800 bp - Peaks get broader and smaller

  15. THE HUMAN GENOME

  16. What’s in a genome? • Genes that code for proteins – 2-3% - contain Open Reading Frame (ORF) beginning with start and stop codons • Many genes have multiple copies or have several closely related ‘family’ members • Regions coding for structural RNA (not proteins)– eg ribosomal RNA, tRNA • Regulatory regions – binding regions for regulatory proteins, transcription factors

  17. Moderately Repetitive DNA • Functional • Gene families eg globin, actin • Gene family arrays eg histone genes, rRNA genes (250 copies), tRNA genes • Without known function • Short interspersed elements (SINES) eg Alu • 200-300 bp long, 100,000s of copies, 13% • Long interspersed elements (LINES) • 1-5 kb long 10-10000 copies per genome, 21% • Pseudogenes

  18. Highly repetitive DNA • About 15% of genome • Minisatellites (Variable number tandem repeats (VNTR) • Repeats of 14-500 bp segments • scattered throughout genome, number of repeats varies on different chromosomes • Microsatellites (Short tandem repeat polymorphisms (STRP) • Regions up to 2-5 bp repeated many times 10-30 copies • Hundreds of kb long • Eg heterochromatin • Telomeres • 6 bp repeat • 250 – 1000 repeats at the end of each chromosome

  19. The race to sequence the human genome • 3 billion bases in the human genome • In 22 pairs of chromosomes + 2 sex chromosomes • Only about 30,000 genes

  20. 2 competing approaches • Hierarchical method • Adopted by the publicly funded Human Genome Project • Sequence of 12 individuals • Whole genome shotgun (WGS) method • Adopted by Celera, a for-profit company • Sequence of 1 individual

  21. Craig Venter • Founder of Celera • Applied whole genome shotgun sequencing method to human genome • Made the first synthetic chromosome

  22. Assignment for next week • You will be working in 4 groups of 4-5. Explain to the class (10-15 min): • Group 1and 2 – explain the following terms related to genome sequencing: 1: mapping, STSs and ESTs, coverage, contigs, golden tiling path, 2: library, BACs, finishing, annotation • Group 3 – explain the hierarchical approach • Group 4 – explain the whole genome shotgun approach

More Related