CHAPTER 8: NUCLEIC ACID DNA SEQUENCING

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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.

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Nội dung Text: CHAPTER 8: NUCLEIC ACID DNA SEQUENCING

INTERNATIONAL UNIVERSITY SCHOOL OF BIOTECHNOLOGY CHAPTER 8: NUCLEIC ACID DNA SEQUENCING BIOCHEMISTRY 1
Learning objectives To understand how DNA is sequenced using the most common technique automated dideoxynucleotide sequencing 2
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 3
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. 4
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. 5
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. 6
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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. 8
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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
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GENOMIC DNA Genomes come in a variety of sizes. Viruses, which cannot live without a host cell, have the smallest genomes, while higher-order organisms such as plants and animals have genomes that are billions of bases long. Like genomes, individual genes can vary greatly in size, from several hundred bases to millions of bases . 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. 14
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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
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GENOMIC DNA The dideoxy DNA sequencing procedure was invented by Frederic Sanger and his colleagues in 1977. With a few improvements, this method is still used today. This elegant procedure, which can be 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
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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 size using capillary electrophoresis. Finally, the DNA code is read by a computer, which displays the data for scientists to use. 20