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

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

6. Concepts of Genetics 7th Ed, Klug and Cummings
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 bp are not reliable, also limited to about 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
bp long, 100,000s of copies, 13%
Long interspersed elements (LINES)
1-5 kb long copies per genome, 21%
Pseudogenes

18. Highly repetitive DNA About 15% of genome
Minisatellites (Variable number tandem repeats (VNTR)
Repeats of 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 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 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