CHAPTER 8: NUCLEIC ACID DNA SEQUENCING doc

1 CHAPTER 8: NUCLEIC ACID DNA SEQUENCING INTERNATIONAL UNIVERSITY SCHOOL OF BIOTECHNOLOGY BIOCHEMISTRY 2 Learning objectives To understand how DNA is sequenced using the most common technique automated dideoxynucleotide sequencing 3 Content 1. INTRODUCTION 2. GENOMIC DNA 3. DNA PREPARATION 4. SEQUENCING REACTION 5. STRAND SEPARATION 6. PRIMER ANNEALING 7. PRIMER EXTENSION 8. CHAIN TERMINATION 9. PUTTING IT ALL TOGETHER 10. CAPILLARY ELECTROPHORESIS 11. COMPUTER ANALYSIS 12. PROCEDURE SUMMARY 13. CONCLUSION 4 INTRODUCTION Automated DNA sequencing is a core research tool used by almost every research biochemistry lab. It is used to determine the sequence of DNA, or the genetic code, that serves as the blueprint of life for every organism on Earth. 5 INTRODUCTION Nucleic acid sequencing is a relatively late arrival for the sequencing of biological macromolecules—for up until the late 1980s protein sequencing was the primary tool for obtaining coding information found in the molecules of life. Protein sequencing is a slow and expensive endeavor, and it could easily take a year or more to sequence a protein of 500 amino acids. 6 INTRODUCTION Today the sequence of a protein can be determined from DNA analyses in just a few days. Because of the straightforward and repetitive nature of the procedure, the sequencing itself is typically performed in centralized facilities where automated machines carry out the reactions and data analysis. 7 8 GENOMIC DNA Since it was discovered that DNA is the material in the cell that carries our genetic information, understanding DNA has become a primary focus of genetic research. Our chromosomes, or genome, consist of neatly wound strands of DNA. All living organisms, from bacteria to human beings, contain DNA in each of their cells. Each cell contains the entire genetic code for that organism. 9 10 [...]... of DNA are called exons 14 15 GENOMIC DNA Introns, which are intervening stretches of DNA that are not fully understood, make up the rest of the gene The largest human gene, dystrophin, a muscle protein implicated in muscular dystrophy, is 2.4 million bases in length Viral and bacterial DNA sequences, which do not contain introns, are typically the shortest genes 16 17 GENOMIC DNA The dideoxy DNA sequencing. .. fully automated, allows large sequencing centers to read over 1,000 bases of DNA sequence per second, a feat which now allows scientists to sequence even large genomes within the span of years, rather than decades 18 19 GENOMIC DNA First, DNA has to be extracted from the cells of the organism being studied The sequencing reaction is then performed on the DNA, and the sequenced DNA strands are sorted by... bacterium divides, the DNA vector placed inside is also copied In this way, the target DNA can be multiplied exponentially Each of the copied DNA pieces is called a clone 29 30 SEQUENCING REACTION The sequencing reaction itself consists of four steps, which will be covered in detail in this section First, the double-stranded DNA is separated into single strands, and a small starter piece of DNA called a primer... proteins The DNA can be selectively removed from this soup by precipitating it and DNAbinding proteins can be cleaned away 22 23 24 DNA PREPARATION Very large pieces of DNA, such as whole chromosomes or genomes, are cut into smaller pieces and stored in vectors (plasmids), which are larger pieces of DNA with the ability to be reproduced when placed in host cells such as bacteria 25 26 27 28 DNA PREPARATION... to one of the strands, called the template strand 31 32 33 SEQUENCING REACTION In the extension step, a new DNA strand is made that is complementary to the template strand Starting at the primer, DNA polymerase uses the template strand as a guide to recreate the second DNA strand 34 35 SEQUENCING REACTION The termination step is the key to the sequencing reaction Strand extension is halted by the incorporation... capillary electrophoresis Finally, the DNA code is read by a computer, which displays the data for scientists to use 20 21 DNA PREPARATION Before it can be sequenced, DNA needs to be purified from cells First, the cells and their nuclei are broken open This can be accomplished by mechanical methods, such as grinding, or by chemical methods that break apart cell membranes The DNA floating around in this “soup”...GENOMIC DNA DNA consists of just four building blocks, or nucleotides These four building blocks, known by their abbreviations A, T, G and C are used as the “alphabet” to write our genetic code All the instructions needed to build our bodies are encoded using just these four letters 11 12 13 GENOMIC DNA Genomes come in a variety of sizes Viruses, which... incorporation of a dye-labeled terminator nucleotide, which identifies the base at the position where strand extension stopped When many strand termination reactions are performed together, each of the bases in a DNA strand can be identified 36 . 1 CHAPTER 8: NUCLEIC ACID DNA SEQUENCING INTERNATIONAL UNIVERSITY SCHOOL OF BIOTECHNOLOGY BIOCHEMISTRY 2 Learning objectives To understand how DNA is sequenced using. Earth. 5 INTRODUCTION Nucleic acid sequencing is a relatively late arrival for the sequencing of biological macromolecules—for up until the late 1980s protein sequencing was the primary. 19 20 GENOMIC DNA First, DNA has to be extracted from the cells of the organism being studied. The sequencing reaction is then performed on the DNA, and the sequenced DNA strands are sorted