Dna sequencing

1. Dna sequencing Aaron Baker and John Sartor

2. What is dna sequencing? • It is a method used to understand how DNA is sequenced using the most common technique automated dideoxynucleotide sequencing. • Nucleic acid sequencing is relatively new • (original) Protein sequencing is slow and difficult. (up until the 1980’s ) • Automated DNA sequencing is rapid and efficient (a few days) • Is a core research tool used by almost all labs • The dideoxy DNA sequencing process was invented by Fredric Sanger and his collogues in 1977 (this method is still used today) and is fully automated. • It is also called chromosome walking • Was used to find the Human Genome

3. Dna preperation • Once DNA has been extracted the strand are sorted by size using capillary electrophoresis. • The DNA needs to be purified from the cells. This is done by precipitating them out. • Large pieces of DNA are cut into smaller pieces and stored in vectors. • Vectors are larger pieces of DNA that have the ability to be reproduced in host cells. Such as bacteria! • These bacteria are then placed in culture medium and allowed to multiply.

4. Sequencing reaction • The Sequencing reactions are completed in four steps. • Strand Separation • Primer Annealing • Primer Extension • Chain Termination

5. Strand separation • The DNA must be denatured before it can be sequenced • This is done by heating

6. Primer Annealing • A small single-stranded DNA piece of about 20 bases, called an oligonucleotide, is annealed to the denatured template strand • The oligonucleotide primer must be of complementary sequence to the template strand in order to bind by base-pair interactions Annealing

7. A bacterial DNA polymerase enzyme begins assembling a new DNA chain from the individual nucleotide building blocks, or dNTPs, provided in the reaction mixture. • The nucleotides are added in the order specified by the complementary bases in the template strand. DNA polymerase cannot start copying a template strand without a small piece of DNA to start the extension process. Oligonucleotide extension!! Primer extension

8. Chain Termination • Since the reaction mixture also contains small amounts of each of the 4 dideoxynucleotides or “ddNTPs each of the four ddNTPs is labeled with a different dye, which can later be detected using a special laser. • The DNA polymerase occasionally incorporates a labeled dideoxynucleotide into a growing DNA strand.

9. First, the reaction mixture is heated to keep the newly synthesized single strands from annealing with the template DNA strand. The dye-labeled single strands are loaded onto a tiny capillary tube containing a viscous, gel-like material. • An electrical current pulls the negatively charged DNA strands through the capillary. • Shorter DNA strands migrate through the gel material more quickly, and come out the bottom of the capillary first, while longer strands become tangled in the gel material and take longer to emerge out the bottom. • As the strands emerge out the bottom of the capillary they pass through a laser beam that excites the fluorescent dye attached to the dideoxynucleotide at the end of each strand. Capillary Electrophoresis Dyed DNA

10. Computer Analysis • The computer displays the information received from the photocell as an electropherogram. It also prints the letter of the appropriate base below each of the signal peaks. • Successive peaks correspond to DNA segments differing in length by one nucleotide, the sequence of peaks reveals the sequence of bases in the original DNA sample.

11. Applications • Entire genomes can be sequenced in this manner. Genome sequencing projects, representing many different organisms, hold the promise of unprecedented advances in industry and medicine. • Microbial genomes may encode enzymes that could help make industrial processes more efficient. Human genome sequences are helping us to better understand human metabolism and disease and may make it easier to treat genetic diseases or design better drugs in the future.

12. Facts! • Understanding DNA has become main focus of genetic research • All organisms contain DNA and contains all genetic code for that organism • Consist of four building blocks (nucleotides A,C,T,G) • Genomes come in a variety of sizes. • Viruses have the smallest genomes and higher-order organisms such as plants and animals have genomes that are billions of bases long. • The average human gene is about 3,000 bases long, although only about 1,000–2,000 bases actually encode protein. These protein-encoding stretches of DNA are called exons. • Introns, which are intervening stretches of DNA that are not fully understood, make up the rest of the gene.