Genome Project - Primer on molecular genetics - 1992

Primer on Molecular Genetics1DOE Human Genome ProgramPrimer on Molecular GeneticsDate Published: June 1992U.S. Department of EnergyOffice of Energy ResearchOffice of Health and Environmental ResearchWashington, DC 20585The "Primer on Molecular Genetics" is taken from the June 1992 DOE HumanGenome 1991-92 Program Report. The primer is intended to be an introduction tobasic principles of molecular genetics pertaining to the genome project.Human Genome Management Information SystemOak Ridge National Laboratory1060 Commerce ParkOak Ridge, TN 37830Voice: 865/576-6669Fax: 865/574-9888E-mail: bkq@ornl.gov2ContentsPrimer onMolecularGeneticsIntroduction ............................................................................................................. 5DNA...............................................................................................................................

6Genes ............................................................................................................................ 7Revised and expandedby Denise Casey(HGMIS) from theprimer contributed byCharles Cantor andSylvia Spengler(Lawrence BerkeleyLaboratory) andpublished in theHuman Genome 1989–90 Program Report.Chromosomes ............................................................................................................... 8Mapping and Sequencing the Human Genome ...................................... 10Mapping Strategies .....................................................................................................Genetic Linkage Maps ............................................................................................Physical Maps .........................................................................................................Low-Resolution Physical Mapping......................................................................Chromosomal map .........................................................................................cDNA map ......................................................................................................High-Resolution Physical Mapping .....................................................................Macrorestriction maps: Top-down mapping ...................................................Contig maps: Bottom-up mapping ..................................................................111113141414141617Sequencing Technologies ...........................................................................................Current Sequencing Technologies .........................................................................Sequencing Technologies Under Development .....................................................Partial Sequencing to Facilitate Mapping, Gene Identification ...............................18232424End Games: Completing Maps and Sequences; Finding Specific Genes ..................

25Model Organism Research .............................................................................. 27Informatics: Data Collection and Interpretation ..................................... 27Collecting and Storing Data ........................................................................................ 27Interpreting Data ......................................................................................................... 28Mapping Databases ....................................................................................................

29Sequence Databases .................................................................................................. 29Nucleic Acids (DNA and RNA) ................................................................................ 29Proteins .................................................................................................................. 30Impact of the Human Genome Project ....................................................... 30Glossary... .............................................................................................................. 3234IntroductionThe complete set of instructions for making an organism is called its genome. Itcontains the master blueprint for all cellular structures and activities for the lifetime ofthe cell or organism.

Found in every nucleus of a person’s many trillions of cells, thehuman genome consists of tightly coiled threads of deoxyribonucleic acid (DNA) andassociated protein molecules, organized into structures called chromosomes (Fig. 1).Fig. 1. The Human Genome at Four Levels of Detail. Apart from reproductive cells (gametes) andmature red blood cells, every cell in the human body contains 23 pairs of chromosomes, each apacket of compressed and entwined DNA (1, 2).

Each strand of DNA consists of repeatingnucleotide units composed of a phosphate group, a sugar (deoxyribose), and a base (guanine,cytosine, thymine, or adenine) (3). Ordinarily, DNA takes the form of a highly regular doublestranded helix, the strands of which are linked by hydrogen bonds between guanine and cytosineand between thymine and adenine. Each such linkage is a base pair (bp); some 3 billion bpconstitute the human genome.

The specificity of these base-pair linkages underlies the mechanismof DNA replication illustrated here. Each strand of the double helix serves as a template for thesynthesis of a new strand; the nucleotide sequence (i.e., linear order of bases) of each strand isstrictly determined. Each new double helix is a twin, an exact replica, of its parent. (Figure andcaption text provided by the LBL Human Genome Center.)5Primer onMolecularGeneticsIf unwound and tied together, the strands of DNA would stretch more than 5 feet butwould be only 50 trillionths of an inch wide.

For each organism, the components of theseslender threads encode all the information necessary for building and maintaining life,from simple bacteria to remarkably complex human beings. Understanding how DNAperforms this function requires some knowledge of its structure and organization.DNAIn humans, as in other higher organisms, a DNA molecule consists of two strands thatwrap around each other to resemble a twisted ladder whose sides, made of sugar andphosphate molecules, are connected by “rungs” of nitrogen-containing chemicals calledbases.

Each strand is a linear arrangement of repeating similar units called nucleotides,which are each composed of one sugar, one phosphate, and a nitrogenous base (Fig.2). Four different bases are present in DNA—adenine (A), thymine (T), cytosine (C), andguanine (G). The particular order of the bases arranged along the sugar-phosphatebackbone is called the DNA sequence; the sequence specifies the exact genetic instructions required to create a particular organism with its own unique traits.Fig.

2. DNA Structure.The four nitrogenousbases of DNA arearranged along the sugarphosphate backbone in aparticular order (the DNAsequence), encoding allgenetic instructions for anorganism. Adenine (A)pairs with thymine (T),while cytosine (C) pairswith guanine (G). The twoDNA strands are heldtogether by weak bondsbetween the bases.A gene is a segment ofa DNA molecule (ranging from fewer than1 thousand bases toseveral million), locatedin a particular position ona specific chromosome,whose base sequencecontains the informationnecessary for proteinsynthesis.Phosphate MoleculeDeoxyriboseSugar MoleculeNitrogenousBasesTACGCGTAWeak BondsBetweenBasesSugar-PhosphateBackbone6The two DNA strands are held togetherby weak bonds between the bases oneach strand, forming base pairs (bp).Genome size is usually stated as the totalnumber of base pairs; the human genomecontains roughly 3 billion bp (Fig.

3).Each time a cell divides into two daughtercells, its full genome is duplicated; forhumans and other complex organisms,this duplication occurs in the nucleus.During cell division the DNA moleculeunwinds and the weak bonds betweenthe base pairs break, allowing the strandsto separate. Each strand directs thesynthesis of a complementary newstrand, with free nucleotides matching upwith their complementary bases on eachof the separated strands.

Strict basepairing rules are adhered to—adenine willpair only with thymine (an A-T pair) andcytosine with guanine (a C-G pair). Eachdaughter cell receives one old and onenew DNA strand (Figs. 1 and 4). Thecell’s adherence to these base-pairingrules ensures that the new strand is anexact copy of the old one. This minimizesthe incidence of errors (mutations) thatmay greatly affect the resulting organismor its offspring.GenesEach DNA molecule contains many genes—the basic physical and functional units ofheredity. A gene is a specific sequence of nucleotide bases, whose sequences carry theinformation required for constructing proteins, which provide the structural components ofcells and tissues as well as enzymes for essential biochemical reactions.

The humangenome is estimated to comprise at least 100,000 genes.Human genes vary widely in length, often extending over thousands of bases, but onlyabout 10% of the genome is known to include the protein-coding sequences (exons) ofgenes. Interspersed within many genes are intron sequences, which have no codingfunction. The balance of the genome is thought to consist of other noncoding regions(such as control sequences and intergenic regions), whose functions are obscure.