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Molecular diagnostics: fundamentals, methods, and clinical applications. by Lela Buckingham. Print book. English. Third edition. Philadelphia: F.A. Davis. Buckingham_Molecular Diagnostics-Fundamentals Methods and Clinical Applications - Ebook download as PDF File .pdf), Text File .txt) or read book online. As new scientific information becomes available through basic and clinical research Molecular diagnostics: fundamentals, methods, and clinical applications /.
Sticky ends can be converted to blunt ends using DNA polymerase to extend the recessed strand in a sticky end. One multi-subunit prokaryotic enzyme is responsible for the synthesis of all types of RNA in the prokaryotic cell. Takai D. The complementary strands could separate and serve as guides of templates for producing like strands. Two types of factors are responsible for regulation of RNA synthesis: The role of sigma factor is to guide the complete enzyme to the proper site of initiation on the DNA. Many of these species are only present in virally infected cells or after introduction of foreign nucleic acid by transformation.
If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you. This exceptional resource introduces the fundamentals of nucleic acid biochemistry as well as the advanced concepts integral to professional practice in today's laboratories.
With a focus on the application of molecular concepts to diagnose diseases, the 2nd Edition reflects the many advances in this rapidly developing field. Discussions of general diagnostic procedures reflect the continuing emergence of new diagnostic technologies to prepare you to meet the challenges of the future.
It would serve the multi- purpose of a textbook and a reference guide as well as troubleshooter. I can imagine it would be equally admired by the educator, student and scientist. Stp 1 Aug 22, Join the membership for readers Aug 22, Similar documents. Stem Cell Therapy in Heart Diseases: A History of Happiness: Successfully reported this slideshow.
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WordPress Shortcode. Published in: Full Name Comment goes here. Are you sure you want to Yes No. This product is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America Last digit indicates print number: Christa Fratantoro Manager of Content Development: Deborah Thorp Developmental Editor: The author s and publisher have done everything possible to make this book accurate, up to date, and in accord with accepted standards at the time of publication.
The author s , editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of the book.
Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised always to check product information package inserts for changes and new information regarding dose and contraindications before administering any drug.
Caution is especially urged when using new or infrequently ordered drugs. Molecular diagnostics: Includes bibliographical references and index.
Molecular diagnosis. Flaws, Maribeth. Molecular Diagnostic Techniques—methods. Nucleic Acids—analysis. QU 58 Bm ] RB B83 For those organizations that have been granted a photocopy license by CCC, a separate system of payment has been arranged. The fee code for users of the Transactional Reporting Service is: To my family, friends, teachers, students, and colleagues, I am forever grateful for your guidance, support, and encouragement.
Programs that educate clinical laboratory professionals have had to incorporate molecular-based diagnostic testing into their curricula just as rapidly despite a lack of formal resources. This textbook was written to address these concerns. The textbook explains the principles of molecular-based tests that are used for diagnostic purposes. Examples of applications of molecular-based assays are included in the text as well as case studies that illustrate the use and interpretation of these assays in patient care.
This textbook is also appropriate for students in other health-related disciplines who have to understand the purpose, principle, and interpretation of molecular-based diagnostic tests that they will be ordering and assessing for their patients.
Practitioners who are performing and interpreting these assays can use this text as a resource for reference and trouble-shooting and to drive the implementation of additional molecular-based assays in their laboratory. Educators should contact their F. Tennessee Audrey E. Tennessee Timothy S. Wyoming Lynn R. Florida ix. Tennessee Mary Ellen Koenn. Nebraska Theola N.
Minnesota Jo Ann Wilson. Virginia Susan M. Ohio Mary E.
Ohio Robert D. West Virginia Huey-Jen Lin. Delaware Teresa S. Elizabeth Morales. Deborah Thorp. Davis who helped get this book from the idea stage to hard copy. Health Professions. We would also like to thank all of the reviewers who gave their time to read and comment on the chapters as they were being developed.
Elizabeth Zygarewicz. Christa A. Davis who was involved in this project. Sam Rondinelli. Developmental Associate. Walther Flemming.
The information in the DNA storage system is based on the order or sequence of nucleotides in the nucleic acid polymer. Miescher also observed that most of the nonnuclear cell components could be lysed away with dilute hydrochloric acid. Effective prevention and treatment of disease will result from the analysis of these sequences in the clinical laboratory. Protein analysis and that of carbohydrates and other molecular species remain. It precipitated upon the addition of acid and redissolved in alkali.
Of course. Molecular techniques are. In Just as computer information storage is based on sequences of 0 and 1. Miescher published a paper on nuclein. Linear assembly of the nucleotides makes up one strand of DNA. We now know that the purpose of DNA. Nitrogen bases are attached to a deoxyribose sugar. In the clinical molecular laboratory. Extraction of these with alkali yielded the same substance isolated from the intact cells.
It is assembled in units or nucleotides that are composed of a phosphorylated ribose sugar and a nitrogen base. Nucleic acids offer several characteristics that support their use for clinical purposes. There are four nitrogen bases that make up the majority of DNA found in all organisms in nature. The term. In his writings. He later isolated a similar viscous material from salmon sperm and noted: The molecular biology laboratory.
He found that he could extract a viscous substance from these cells. Addition of extract of pig stomach a source of pepsin to dissolve away contaminating proteins resulted in a somewhat shrunken but clean preparation of nuclei. These are adenine. If the ribose sugar is phosphorylated.
B Two hydrogen bonds form between adenine and thymine. Nitrogen bases are planar carbon-nitrogen ring structures. Bases with a double ring guanine. Adenosine with three phosphates is adenosine triphosphate ATP. For example. The hydroxyl group on the third carbon is important for forming the phosphodiester bond that is the backbone of the DNA strand.
The four common nitrogen bases in DNA are adenine. Nucleotides The four nucleotide building blocks of DNA are molecules of about kd. Nitrogen bases with a single ring thymine. The phosphodiester backbones of the two nucleic acid chains form the helix. Amine and ketone substitutions around the ring as well as the single or double bonds within the ring distinguish the four bases that comprise the majority of DNA Fig.
Nitrogen bases are oriented toward the center where they hydrogen-bond with homologous bases to stabilize the structure. Nucleotides can be converted to nucleosides by hydrolysis. Three hydrogen bonds form between guanine and cytosine. A nitrogen base bound to an unphosphorylated sugar is a nucleoside. Adenosine A. Numbering of the positions in the nucleotide molecule starts with the ring positions of the nitrogen base.
Stress and torsion can throw the double helix into a Z-form. ZDNA is a left-handed helix with 12 bp per turn and altered geometry of the sugar-base bonds. Guanine forms three hydrogen bonds with cytosine. Z-DNA has been observed in areas of chromosomes where the DNA is under torsional stress from unwinding for transcription or other metabolic functions. As DNA is polymerized. The carbons of the ribose sugar are numbered 1 to 5. In this way the parental DNA strand can be replicated without loss of the nucleotide order.
Changes can nated C or N 1. Watson-Crick base pairing purine: The molecule without the phosphate group is the nucleoside. Changes such as methylation of nitrogen bases have biological consequences for gene function and are intended in nature.
Base pairs bp other than A: T and G: C or mismatches. Dehydrated DNA takes the A-form with about 11 bp per turn and the center of symmetry along the outside of the helix rather than down the middle as it is in the B-form.
Both A. Adenine forms two hydrogen bonds with thymine see Fig. It is composed of deoxyribose covalently bound at its number 1 carbon to the nitrogen base. The base carbons are numbered 1 through 9. The sugar carbons are numbered 1 to 5. The phosphate group on the 5 carbon and the hydroxyl group on the 3 carbon form phosphodiester bonds between bases.
Thymine and cytosine have pyrimidine ring structures. The nitrogen bases. These changes can affect gene function as well. The bases are positioned such that the sugar-phosphate chain that connects them sugar-phosphate backbone is oriented in a spiral or helix around the nitrogen bases see Fig.
An analog of guanosine. A nucleic acid chain grows by the attachment of the 5 phosphate group of an incoming nucleotide to the 3 hydroxyl group of the last nucleotide on the growing chain Fig.
The anticancer drugs. Azidothymidine Retrovir. In the laboratory. DNA found in nature is mostly double-stranded. We refer to DNA as oriented in a 5 to 3 direction. The techniques used for these procedures will be discussed in later chapters.
They are in antiparallel orientation with the 5 end of one strand at the 3 end of the other Fig. The replication apparatus is designed to copy the DNA strands in an orderly way with minimal errors before each cell division. The new double helix consists of the template strand and the new daughter strand oriented in opposite directions from one another. The order of nucleotides is maintained because each strand of the parent double helix is the template for a newly replicated strand.
The resulting double strand. DNA polymerase. It is important that this information. The sequences of the two strands that form the double helix are complementary. The template strand is copied in the opposite 3 to 5 direction. As DNA synthesis proceeds in the 5 to 3 direction. Identical sequences will not hybridize with each other. The double helix can also be penetrated by intercalating agents. As Watson and Crick predicted.
Denaturing agents such as formamide and urea displace the hydrogen bonds and separate the two strands of the helix. In the process of replication. The two regions of the helix formed by the backbones are called the major groove and minor groove. Every cell in a multicellular organism or in a clonal population of unicellular organisms carries the same genetic information. The new strand is elongated by hydrogen bonding of the. The enzyme reads the template in the 3 to 5 direction.
Another requirement for DNA synthesis is the availability of the deoxyribose 3 hydroxyl oxygen for chain growth. Upon the description of the double helix. An Overview Historical Highlights Before the double helix was determined. Primase must work repeatedly on the lagging strand to prime synthesis of each Okazaki fragment. They could differentiate true semiconservative replication from dispersive replication by demonstrating that approximately half of the DNA double helices from the next generation grown in normal nitrogen were14N: Erwin Chargaff10 made the observation that the amount of adenine in DNA corresponded to the amount of thymine and the amount of cytosine to the amount of guanine.
The fragments chased into larger pieces with time. The question arises as to how one of the strands of the duplex can be copied in the same direction as its complementary strand that runs antiparallel to it.
The duplicated helix will ultimately consist of one template strand and one new strand. These small fragments. In their experiments. Historical Highlights A few years after solution of the double helix. The two strands of the parent helix are not copied in the same way. These molecules were of intermediate density to the ones from bacteria grown only in 14N or 15N. DNA undergoing active replication can be observed by electron microscopy as a forked structure. Watson and Crick. The complementary strands could separate and serve as guides of templates for producing like strands.
A preceding base must be present to provide the hydroxyl group. Watson proposed that the steps in the ladder of the double helix were pairs of bases. Orthophosphate is released with the formation of a phosphodiester bond between the new nucleotide and the last nucleotide of the growing chain.
The 5 to 3 strand copied in a discontinuous manner is the lagging strand13 Fig. After shifting the bacteria into a medium of normal nitrogen 14N. This means that DNA cannot be synthesized de novo.
This base is provided by another enzyme component of the replication apparatus. Sylvy Kornberg. The lagging strand is read discontinuously. Both strands are read in the 3 to 5 direction. Helicase activity in the replisome unwinds and untangles the DNA for replication.
In vivo. Primase activity is required throughout the replication process to prime the discontinuous synthesis on the lagging DNA strand.
Rnase H. The other two polymerases were responsible for repair of gaps and discontinuities in previously synthesized DNA. Julius Adler. Bessman reported on an extract of E. Once DNA is primed and synthesized. Separate polymerase proteins add incoming nucleotides to the growing DNA strands of the replication fork. This ratio was not affected by the proportion of free nucleotides added to the initial reaction.
Robert Lehman. During the next 3 years. Two of the 10 subunits of the holoenzyme are catalytic DNA polymerizing enzymes. The details of synthesis of the lagging strand are not yet clear. At the time it was difficult to determine whether the new DNA was a copy of the input molecule or an extension of it. Any source of preformed DNA would work.
Arthur Kornberg. The holoenzyme works along with a larger assembly of proteins required for priming. The E. An Overview Table 1. Thermotoga maritima. T4 bacteriophage Eukaryotes Various viruses E.
Pol E. One purpose of the exonuclease function in the various DNA polymerases is to protect the sequence of nucleotides. This is important in the laboratory where prokaryote polymerases are used extensively to copy DNA from many different sources.
The catalytic domain of E. Several cofactors and accessory proteins are required to unwind the template helix green. T4 pol Pol. Rad30 Rad 6. Copying errors will result in base changes or mutations in the. It also explains how a bacterial polymerase can replicate DNA from diverse sources. The large fragment without the exonuclease activity Klenow fragment has been used extensively in the laboratory for in vitro DNA synthesis.
T7 bacteriophage Mitochondria E. Pol V Rev1. At a nick. The three polymerases resemble prokaryotic enzymes. Two polymerase protein complexes. During DNA synthesis.
This concurrent synthesis and hydrolysis then move the nick in one strand of the DNA forward in an activity called nick translation.
This enzyme will add nucleotides to the end of a DNA strand in the absence of hydrogen base pairing with a template. DNA polymerase tries a second time. The polymerization and hydrolysis will proceed for a short dis- tance until the polymerase is dislodged.
Advanced Concepts After replication. This activity degrades duplex DNA from the 5 end and can also cleave diester bonds several bases from the end of the chain. During DNA replication. Another type of DNA polymerase. The 3 to 5 exonuclease function is required to assure that replication begins or continues with a correctly base-paired nucleotide.
The resulting labeled products are used for DNA detection in hybridization analyses. It is important for removing lesions in the DNA duplex such as thymine or pyrimidine dimers. If these structures are not removed.
A opposite C instead of T on the template in the primer sequence before beginning polymerization. The nick can then be reclosed by DNA ligase. The enzyme will remove a mismatch for example. Nick translation is often used in vitro as a method to introduce labeled nucleotides into DNA molecules.
Advanced Concepts Like prokaryotes. A fourth polymerase. DNA polymerases play a central role in modern biotechnology. X based on sequence structure.
They are key tools of recombinant DNA technology.
Endonucleases break the sugar phosphate backbone of DNA at internal sites. After polymerization.
Chemical manipulation of the amino acid structure of these enzymes produces polymerases with characteristics that are useful in the laboratory. Some of these enzymes. Some prefer single-stranded and some prefer double-stranded DNA. As the chemical structure of DNA is the same in all organisms. A host of enzymes performs these and other functions during various stages of the cell cycle. New information as to the nature of these enzymes indicates that polymerases can participate in cohesion holding together of sister chromatids to assure proper recombination and segregation of chromosomes.
DNA polymerase extends the 3 end of a nick in double-stranded DNA with newly synthesized strand gray while digesting the original strand from the 5 end. In addition. Repair endonucleases function at areas of distortion in the DNA duplex such as baseless apurinic or apyrimidic sites on the DNA backbone. These include altered processivity staying with the template longer to make longer products. Polymerases in the A and B family are most useful for biotechnological engineering.
These enzymes were originally isolated from bacteria where they function as part of a primitive defense system to cleave foreign DNA entering the bacterial cell. The site of cleavage of the DNA substrate can be over bp from this binding site. Type II restriction enzymes have been found in almost all prokaryotes. SmaI from Serratia marcescens Sbb and so forth. The single-strand ends can hybridize with complementary ends on other DNA fragments.
Like type I. Recognition sites for these enzymes are asymmetrical. Type II restriction enzymes are those used most frequently in the laboratory. Because of their ability to form hydrogen bonds with complementary overhangs. Restriction enzymes are frequently used in the clinical laboratory.
They bind as simple dimers to their symmetrical DNA recognition sites. Advanced Concepts Restriction enzyme recognition sequences in the DNA are generally areas of bilateral rotational symmetry around an axis perpendicular to the DNA helix. These enzymes do not have inherent methylation activity in the same molecule as the nuclease activity.
Type I restriction enzymes have both nuclease and methylase activity in a single enzyme. DNA Chapter 1 13 polymer at the sugar-phosphate backbone. It recognizes the site: Some enzymes cut the duplex with a staggered separation at the recognition site.
The enzymes bind to the recognition site. Restriction enzymes are named for the organism from which they were isolated. Although all type II restriction enzymes work with bilateral symmetry.
BamHI was isolated from Bacillus amyloliquefaciens H. The enzymes then cleave the DNA backbone at sites symmetrically located around the same twofold axis. An example of a type I enzyme is EcoK from E. It is one of the few enzymes with 5 exonuclease activity. Sticky ends can be converted to blunt ends using DNA polymerase to extend the recessed strand in a sticky end. Its activity is optimal on long single-stranded ends.
These operations require reunion of the DNA backbone after discontinuous replication on the lagging strand. Nuclease Bal31 from Alteromonas espejiani can degrade single. The collection of fragments generated by digestion of a given DNA fragment.. Restriction enzymes can be used for mapping a DNA fragment.
DNA ligases are more efficient in joining DNA ends and have been found in a wide variety of bacteria. This is not true for sticky ends. Exonuclease VII from E. Its existence was predicted by the observation of replication. Exonuclease I from E. An Overview Advanced Concepts The advantage of blunt ends for in vitro recombination is that blunt ends formed by different enzymes can be joined. Exonuclease III from E. DNA duplex at the same place on both strands. It also has some endonuclease activity.
RNA ligase. These enzymes are used. The ability to convert open or nicked circles of DNA to closed circles. Because its activity at 20oC is slow enough to control with good resolution.
Exo III removes nucleotides from blunt ends. DNA Ligase DNA ligase catalyzes the formation of a phosphodiester bond between adjacent 3 -hydroxyl and 5 -phosphoryl nucleotide ends. These ends can be rejoined as well. It was used extensively in early RNAse protection assays of gene expression. It has some endonuclease activity on duplex DNA. Also note the complementary overhangs in Figure Blunt ends top or noncomplementary overhangs center are joined less efficiently than complementary overhangs bottom.
Although not yet available when ligase activity was being studied. These ends have naturally occurring single-stranded overhangs.
It is used in the laboratory to remove nucleic acid from crude extracts and also for analysis of chromatin structure. Most DNA isolation procedures are designed to minimize both endonuclease and exonuclease activity during DNA isolation.
Gobind Khorana showed that short synthetic segments of DNA with singlestrand complementary overhangs joined into larger fragments efficiently. Release of DNA for transcription. The joining reaction required the chance positioning of two adjacent ends and was. It is also used for nuclease mapping techniques. Helicases DNA in bacteria and eukaryotes does not exist as the relaxed double helix as shown in Figure but as a series of highly organized loops and coils. Micrococcal nuclease digests single.
Although this enzyme can digest duplex DNA. These functions are carried out by a series of enzymes called helicases. Because it leaves doublestranded regions intact. DNA Chapter 1 15 Historical Highlights The initial analysis of the joining reaction was performed with physically fractured DNA helices that had no homology at their ends.
It also has endonuclease capability to hydrolyze single-stranded regions such as gaps and loops in duplex DNA. A better substrate for the enzyme would be ends that could be held together before ligation. As nucleases are natural components of cellular lysates. Formerly called exonuclease II. Although it has no activity at nicks short single-strand gaps in the DNA. DNA pol I from E. An Overview As described with restriction endonucleases.
Topo I can relax supercoils in circular plasmid DNA by nicking one strand of the double helix. When only one backbone is broken a single-strand break or nick. They also separate linked rings of DNA concatamers. Because of their importance in cell replication. In eukaryotes. Most prokaryotic DNA is methylated. These topoisomerase inhibitors bring about cell death by interfering with the breaking and joining activities of the enzymes.
There are two main types of methyltransferases. In contrast. These ends can be digested by exonuclease activity or extended using the intact strand as a template nick translation. Unlike prokaryotic DNA. These enzymes catalyze the transfer of an activated methyl group from S-adenosyl methionine to the 5 position of the cytosine ring producing 5-methyl cytosine.
Advanced Concepts Cytosine methyltransferases are key factors in vertebrate development and gene expression. Topoisomerases interconvert topological isomers or relax supertwisted DNA. DNA as required. Topoisomerases in eukaryotes have activity similar to that in bacteria but with different mechanisms of cutting and binding to the released ends of the DNA.
Methylation is the source of imprinting of DNA.