A Lot More Advanced Biotechnology Tools DNA Sequencing

–dideoxynucleotides ddATP, ddGTP, ddTTP, ddCTP missing O for bonding of next nucleotide terminates the growing chain DNA Sequencing

Sanger method –synthesize complementary DNA strand in vitro –in each tube: “normal” N-bases dideoxy N-bases –ddA, ddC, ddG, ddT DNA polymerase primer buffers & salt

Reading the sequence Load gel with sequences from ddA, ddT, ddC, ddG in separate lanes –read lanes manually & carefully –polyacrylamide gel

Fred Sanger 1978 | 1980 This was his 2nd Nobel Prize!! –1st was in 1958 for the structure of insulin

The Sanger Method: DNAi video tutorial

Advancements in sequencing Fluorescent tagging –no more radioactivity –all 4 bases in 1 lane each base a different color Automated reading

Applied Biosystems, Inc (ABI) built an industry on these machines More Advancements in sequencing  Capillary tube electrophoresis  no more pouring gels  higher capacity & faster

PUBLIC  Joint Genome Institute (DOE)  MIT  Washington University of St. Louis  Baylor College of Medicine  Sanger Center (UK) PRIVATE  Celera Genomics  Big labs!  economy of scale

Automated Sequencing machines  Really BIG labs!

Human Genome Project U.S government project –begun in 1990 estimated to be a 15 year project –DOE & NIH initiated by Jim Watson led by Francis Collins –goal was to sequence entire human genome 3 billion base pairs Celera Genomics –Craig Venter challenged gov’t –would do it faster, cheaper –private company

Different approaches 3. Assemble DNA sequence using overlapping sequences. “map-based method” gov’t method “shotgun method” Craig Venter’s method 1. Cut DNA entire chromosome into small fragments and clone. 2. Sequence each segment & arrange based on overlapping nucleotide sequences. 1.Cut DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments. 2. Cut and clone into smaller fragments.

Shotgun!

Human Genome Project On June 26, 2001, HGP published the “working draft” of the DNA sequence of the human genome (4 years ahead of schedule). Historic Event! –blueprint of a human –the potential to change science & medicine

Sequence of 46 Human Chromosomes 3 billion base pairs 3G of data

Things Are Strange In Here:

Raw genome data

NCBI GenBank Database of genetic sequences gathered from research Publicly available on Web!

Maps of human genes… Where the genes are… –mapping genes & their mutant alleles

Defining a gene… “Defining a gene is problematic because… one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.” – Elizabeth Pennisi, Science 2003 gene polypeptide 1 polypeptide 2 polypeptide 3 protein gene RNA

Types of DNA sequences in the human genome Exons (regions of genes coding for protein, rRNA, tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (about 15%) Alu elements (10%) Simple sequence DNA (3%) Large-segment duplications (5–6%)

And we didn’t stop there…

Genome Sizes and Estimated Numbers of Genes*

What have we found? When you go looking…

…you will certainly find something!

Sing A Silly Song Extolling Automation!

Ethical Questions…

1. You have a familiar history of a terminal genetic disease. A genetic test exists for the disease. Would you take the test to see if you will have the disease?

2. As a condition of your continued employment, your boss wants you to have a genetic screening test and the results sent to the company. Will you get the test?

3. You and your spouse have decided to have a child. You have the resources to pre-determine your child’s gender. Would you do this? 4. What about your child’s intelligence?

5. You discover your favorite food contains genetically modified ingredients. Will you continue to eat the food?

6. As part of a routine medical procedure, your doctor discovers that you have a rare, beneficial variant of a protein that protects you from heart disease. Should your doctor be able to patent the protein? 7. Should you be entitled to any money from the patent?