1 *Chapter 32* Molecular Diagnosis in the Clinical Laboratory Three main areas of hemetopathologic molecular testing: a) Detection of chromosomal

al translocation in hematologic malignancies and inherited hematologic disorders b) Identification of hematologically important infectious diseases c) Monitoring of minimal residual disease after cancer treatment Structure and Function of DNA The central dogma: DNA to RNA to Protein The central dogma in genetics is that information is stored in the DNA is replicated to daughter DNA, transcribed to messenger ribonucleic acid (mRNA), and translated into a functional protein DNA is long-term storage. It is stable, packaged, and inert. RNA is short-term storage. It is unstable and lacks secondary structure. Some RNA has enzymatic activity. Proteins are the 'programs' of the cells. They are the physical manifestations of the abstract information recorded in the genome. DNA at the molecular level DNA is the genetic material of all living cells and of many viruses. DNA is: an alpha double helix of two polynucleotide strands. The genetic code is the sequence of bases on one of the strands. A gene is a specific sequence of bases, which has the information for a particular protein. DNA is self-replicating - it can make an identical copy of itself. Replication allows the genetic information to pass faithfully to the next generation. Replication occurs during the S (= synthesis) stage of interphase just before nuclear division. The chromosomes contain 90% of the cells DNA. 10% is present in mitochondria and chloroplasts. Adenine (A) and Guanine (G) are purine bases Thymine (T) and Cytosine (C) are pyrimidine bases Hydrogen bonds link the complementary base pairs: a) Two between A and T (A=T) b) Three between G and C (GC) A single unit in the chain is a nucleotide. This consists of a phosphate group A pentose sugar (D = DNA; R = RNA) and An organic base (ATGC = DNA; AUGC = RNA) Transcription and translation

RNA Synthesis: Transcription

RNA is an important type of nucleic acid that plays several roles in the production of protein RNA is necessary to carry the instructions of the DNA out of the nucleus and to the ribosomes The genome of any organism contains all the information for making that organism. The information is encoded in various types of genes that are transcribed into 4 types of RNA: a) mRNA - Messenger RNA: Encodes amino acid sequence of a polypeptide b) tRNA - Transfer RNA: Brings amino acids to ribosomes during translation c) rRNA - Ribosomal RNA: With ribosomal proteins, makes up the ribosomes, the organelles that translate the mRNA d) snRNA - Small nuclear RNA: With proteins, forms complexes that are used in RNA processing in eukaryotes

2 Messenger RNA carries the actual code that specifies the amino acid sequence in a polypeptide (protein) Making mRNA starts with a protein encoding gene on a template strand of DNA Step 1: Initiation RNA Polymerase binds to a promoter which is a region of bases that signals the beginning of a gene RNA Polymerase is bound to the TATA box of the promoter by transcription factors The double helix unwinds and is ready to be transcribed Step 2: Elongation RNA Polymerase moves along the protein encoding gene adding new RNA nucleotides in the 5 to 3 direction and complimentary to the DNA template Works at up to 60 nucleotides/second Step 3: Termination RNA Polymerase reaches the terminator region of the protein encoding gene All the enzymes and factors are released The product of these 3 steps is called immature or pre-mRNA RNA Processing Most eukaryotic protein encoding genes contain non-coding segments called introns, which break up the amino acid coding sequence into segments called exons RNA Processing includes modification and splicing Modification At the 5' end, a cap is added consisting of a modified GTP (guanosine triphosphate). This occurs at the beginning of transcription. The 5' cap is used as a recognition signal for ribosomes to bind to the mRNA At the 3' end, a poly(A) tail of 150 or more adenine nucleotides is added. The tail plays a role in the stability of the mRNA Splicing (Intron Removal) The intron loops out as snRNPs (small nuclear ribonucleoprotein particles) bind to form the spliceosome The intron is excised, and the exons are then spliced together Results in mature mRNA

Protein Synthesis: Translation

The language of nucleic acids in translated into the language of proteins Nucleic acids have a 4 letter language Proteins have a 20 letter language Things needed in translation: a) Messenger RNA (mRNA) Synthesized in Transcription Composed of Codons Codons are 3-base sequences of mRNA b) Ribosomes Made of rRNA and protein 2 subunits (large and small) form a 3D groove 2 major sites: P site---holds the growing polypeptide A site---new amino acids enter here c) Transfer RNA (tRNA) Carries amino acids to the ribosome During tRNA charging each tRNA picks up an amino acid from the INP d) Amino Acids There are 20 amino acids, each with a basic structure Amino acids are held together by peptide bonds 3 Steps in translation: 1) Initiation 2) Elongation 3) Termination

3 Step 1: Initiation 5 G-cap of mRNA binds to ribosome Start codon AUG and anticodon with Methionine bind a P site A site is open and ready to receive new tRNAs Step 2: Elongation (Adding New Amino Acids) Codon recognition Peptide bond formation Translocation: ribosome moves along mRNA, aminoacyl tRNA shifts from A site to P site Step 3: Termination A stop codon is reached UAA UAG UGA All parts release Deletion or insertion mutations are most disruptive because they change the reading frame, causing a frame shift Substitution mutations have varied impact on amino acid sequences. Substitutions of 1st or 2nd base in codon almost always changes the amino acid Substitution of 3rd base in codon does not always change the amino acid What causes mutations? Errors in DNA Replication Errors in chromosome crossover in meiosis Mutagens Mutagens are physical or chemical factors that cause mutations UV Radiation and X-Rays Chemicals like DDT Many mutations are harmful and cause the organism to die or function incorrectly. Some mutations are beneficial and help the organism to survive. (Peppered Moths) If mutations are present in gametes, they can be passed on to offspring. This is the driving force of Natural Selection. DNA replication and the cell cycle

Translation, Polypeptides, and Mutations

Normally, the genetic code is translated and the correct protein is formed from a long chain of amino acids. Translation of codons is dependent on the reading frame, or a grouping of codons in a gene transcript. Mutations: Any change in the nucleotide sequence of DNA Mutations can involve large sections of chromosomes or single base pairs Mutations can change the reading frame of a gene transcript

DNA replication
DNA Replication is semi-conservative Each newly synthesized molecule contains 1 parent template strand and 1 new daughter strand Step 1: Initiation Helicase unwinds DNA forming a replication fork Multiple replication forks along a DNA molecule create replication bubbles Step 2: Elongation---Adding New Nucleotides RNA Primase adds a complimentary RNA primer to each template strand as a starting point for replication

4 DNA Polymerase reads the template strand (3 to 5) and adds new complimentary nucleotides (5 to 3) DNA synthesized in the direction of the replication fork is called the leading strand DNA polymerase can only add new nucleotides in the 5 to 3 direction Because of the antiparallel nature of DNA, replication occurs in two directions An RNA primer is laid down on the other strand, and new nucleotides are added 5 to 3 moving away from the replication fork. This is the lagging strand and the segment of DNA produced is called an Okazaki fragment The DNA unwinds some more and the leading strand is extended by DNA polymerase adding more DNA nucleotides. Thus, the leading strand is synthesized continuously. On the top template strand, a new RNA primer is synthesized by primase near the replication fork DNA polymerase adds new DNA. This produces the second Okazaki fragment. Thus, the lagging strand is synthesized discontinously Step 3: Termination A different type of DNA polymerase removes the RNA primer and replaces it with DNA DNA ligase joins the two Okazaki fragments with phosphodiester bonds to produce a continuous chain Each new DNA molecule is rewound by helicase. Each molecule is identical Summary and Other Facts: Leading Strand: 1 primer, 5 to 3 continuous Lagging Strand: multiple primers, 5 to 3 discontinuous In humans, DNA polymerase adds 50 nucleotides/second DNA polymerase can proofread its own work and does excision repair 1 in 10,000 bases are in error After proofreading, rate of mutation is 1 in 10,000,000

Cell cycle

Gap 1-phase The first phase within interphase, from the end of the previous M phase until the beginning of DNA synthesis. It is also called the growth phase. During this phase the biosynthetic activities of the cell, which had been considerably slowed down during M phase, resume at a high rate. This phase is marked by synthesis of various enzymes that are required in S phase, mainly those needed for DNA replication. Duration of G1 is highly variable, even among different cells of the same species. S-phase S-phase starts when DNA synthesis commences; When it is complete, all of the chromosomes have been replicated, i.e., each chromosome has two (sister) chromatids. Thus, during this phase, the amount of DNA in the cell has effectively doubled. It is the longest phase of the cell cycle.

5 G2 -phase G2 phase, which lasts until the cell enters mitosis. Again, significant biosynthesis occurs during this phase, mainly involving the production of microtubules, which are required during the process of mitosis. Inhibition of protein synthesis during G2 phase prevents the cell from undergoing mitosis. Stages of Mitosis 1.Prophase The chromosomes gradually condense and appear as strands that become thicker and shorter; - The nuclear envelope breaks up. 2.Metaphase The chromosomes are condensed; - A mitotic spindle is formed of microtubules; - Microtubules attach to the centromeres on chromosomes and to the centrioles at opposite poles of the cell 3.Anaphase The chromatids separate and move to opposite poles 4.Telophase The chromatids are at opposite poles of the cells - The nuclear envelope is formed 5.Cytokinesis Division of the cytoplasm mediated by actin filament Regulation of cell cycle A major cell cycle regulatory point occurs in late G1 phase and controls progression from G1 to S. This regulatory point was first defined by studies of budding yeast (Saccharomyces cerevisiae), where it is called as START Once cells have passed START, they are committed to enter S phase. In addition to serving as a decision point for monitoring extracellular signals, START is the point at which cell growth is coordinated with DNA REPLICATION and CELL DIVISION Families of cyclins and cyclin dependent kinases The cell cycles of higher eukaryotes are controlled not only by multiple cyclins but also by multiple cdc2 related protein kinases-cdks Active complexes o cyclins and CDKs exert their biological effects by phosphorylating proteins During the G1 phase, a major target of cyclin/CDK complexes is the retinoblastoma protein (pRb). pRb is a growth-suppressing protein whose activity is controlled by whether or not it is phosphorylated pRb When pRb is in the dephosphorylated form, during the G0 phase and early in the G1 phase, it is active pRb exerts its growth-suppressing effects by binding to many cellular proteins, including the transcription factors of the E2F family E2F transcription factors regulate the expression of numerous genes that are expressed during G1, or at the transition from the G1 to the S phase, to initiate DNA replication. pRb that is bound to an E2F transcription factor inhibits the transcription factor's activity. Following phosphorylation by cyclin/CDK complexes, pRb dissociates from E2F, allowing the transcription factor to bind DNA sequences and activate the expression of genes necessary for the cell to enter the S phase. p53 The p53 protein senses DNA damage and can halt progression of the cell cycle in G1 (by blocking the activity of Cdk2 Under normal circumstances p53 levels remain very low due to its interaction

6 with a member of the ubiquitin ligase family called MDM2. The p53 protein is also a key player in apoptosis, forcing "bad" cells to commit suicide. So if the cell has only mutant versions of the protein, it can live on perhaps developing into a cancer. More than half of all human cancers do, in fact, harbor p53 mutations and have no functioning p53 protein. An extreme case of this is Li Fraumeni syndrome, where a genetic a defect in p53 leads to a high frequency of cancer in affected individuals. A genetically engineered adenovirus, called ONYX-015, can only replicate in human cells lacking p53. Thus it infects, replicates, and ultimately kills many types of cancer cells in vitro. Clinical trials are now proceeding to see if injections of ONYX-015 can shrink a variety of types of cancers in human patients. Telomere and Telomerase Telomeres, located at the ends of chromosomes, are key genetic elements involved in the regulation of the cellular aging process. Each time a normal cell divides, telomeres shorten Once telomeres reach a certain short length, cell division halts and the cell enters a state known as replicative senescence or aging. Thus, this shortening of the telomeres effectively serves as a molecular "clock" for cellular aging. When the enzyme telomerase is introduced into normal cells, it can restore telomere length - reset the "clock" - thereby increasing the functional lifespan of the cells. Importantly, it does this without altering the cells' biology or causing them to become cancerous. Telomerase Human telomerase present at very low levels, in most normal cells and tissues, but that during cancer progression, telomerase is abnormally reactivated in all major cancer types. While telomerase does not cause cancer, the continued presence of telomerase enables cancer cells to maintain telomere length, providing them with indefinite replicative capacity. It has been shown in various tumor models that inhibiting telomerase activity results in telomere shortening and causes aging or death of the cancer cell. Development of anti-cancer therapies based on telomerase inhibitors and telomerase therapeutic vaccines Molecular Diagnostic Testing Overview Generalities Exploits the enzymes & processes of DNA replication to make copies of DNA sequence of interest

Variations within different cells The human body contains a huge range of cells ~ over 300 different types. The fastest cycling mammalian cells in culture, crypt cells in the intestinal epithelium, have a cycle time as short as 9 to 10 hours. Cells are most radiosensitive in late M and G2 phases and most resistant in late S phase Some divide often through life, others divide only infrequently and some do not divide at all after birth. The most actively dividing cells are found in areas of the body that receive a lot of wear and tear. (Skin, GIT) Liver tissue is very interesting. It is usually permanent but when massive damage to the liver occurs, the cells can get out of G0 and undergo mitosis to repair the tissue

 Amplicons -> copies Primers or Probes short sequences used to locate specific DNA or RNA sequences within a population of nucleic acids For mRNA or rRNA amplification happens in a process called reverse transcriptase PCR Most test use DNA amplification Contamination Prevention Use UV rays and bleach to induce stand breaks in work surfaces Use Uracil-N-Glycosylase to destroy previously amplified DNA Nucleic Acid Isolation (Extraction) Isolating DNA from clinical specimen Used to test for mutation in patient DNA Used to test for microorganism DNA (detecting infections) DNA is preferred since it is more stable than RNA and easier to isolate Collection Samples can include peripheral blood, bone marrow, tissue biopsy, needle aspiration, cheek swabs Whole blood is collected in an EDTA tube to prevent clotting and inhibit enzymes that may digest DNA White blood cells are separated by detergent and proteinase A high salt solution removes cellular debris leaving DNA in the aqueous solution The high salt solution neutralizes the negative charge of backbone allowing close contact of DNA with one another. Addition of isopropanol precipitates DNA

Wash with 70% ethanol and resuspend in aqueous buffer solution Isolated DNA sample can be stored at -80C DNA can be taken from formalin fixed, paraffin embedded tissue sections Tissue is obtained by microdissection by scraping or laser Tissue is degraded by proteinase K to release DNA Sample is heated to 94C for several minutes to inactivate the proteinase K and degrade other proteins Fresh/Frozen tissue samples can be utilized as well by quickly thawing and mincing the frozen tissue The minced tissue is mixed with an extraction buffer to release DNA and is purified and precipitated as described earlier Isolating RNA from clinical specimen Much more difficult than DNA isolation The isolated RNA contains mRNA, rRNA, tRNA. Large specimen may be needed to obtain adequate amount of mRNA mRNA does not represent all information stored in the DNA, only the genes being expressed. Collection RNA released by cell lysis RNase inhibition with strong chemical agents such as urea or guanidine isothiocyanate Protein and DNA removal by using phenol at pH 4, chloroform and isoamyl alcohol. The chemicals separate DNA and protein in the organic layer while RNA remains in the aqueous phase. RNA resists acidic pH while DNA, carbohydrates, lipids

and proteins are affected by an acidic pH RNA precipitation by addition of salt to neutralize the charge of the phosphoester backbone and ethanol to make the nucleic acid insoluble Amplification of Nucleic Acid by Polymerase Chain Reaction Polymerase chain reaction for amplifying DNA PCR is an enzyme based method for amplifying a target sequence to allow its detection from a small volume of material DNA is first denatured at 95C to separate the strands It is then cooled to 40-60C to allow annealing (binding) of the primer to the target sequence It is then warmed to 72C to promote the Taq polymerase. The Taq polymerase attaches to the primers and extend the strand to synthesize the new DNA The cycle is then repeated A thermocycler is used to accurately produce and monitor rapid temperature changes Primer annealing accounts for PCR specificity but primers anneal to nonidentical regions if annealing temperature is too low. PCR controls include +, - and no DNA controls - control contains DNA that lacks the sequence of interest + control contains DNA that has the sequence of interest No DNA indicates if there is DNA contamination. Ex a band formed in no DNA means that sample is contaminated.

Reverse Transcription polymerase chain reaction for amplifying RNA In RT PCR, reverse transcriptase enzyme produces complementary DNA (cDNA) from mRNA Produce an RNA-CDNA hybrid by utilizing reverse transcriptase and a specialized primer called oligo(dT) The primer anneals to the polyA tail on the 3 end of the adenine nucleotides and the reverse transcriptase recognizes the hydroxyl group on the last nucleotides of the primer and reads the mRNA template strand and then adds the correct complementary deoxyribo nucleotides Heat denaturation separated the mRNA from the cDNA strand so that the can act as a template for replication by DNA polymerase. The cDNA is then amplified as in DNA based PCR using primers specific for the sequence of interest.

9 Detection of Amplified DNA Gel electrophoresis Gel electrophoresis is a method that separates macromolecules either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, posses ionisable groups and, therefore, at any given pH, exist in solution as electically charged species either as cations (+) or anions (-). Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode. Separation of large (macro) molecules depends upon two forces: charge and mass. When a biological sample, such as proteins or DNA, is mixed in a buffer solution and applied to a gel, these two forces act together. The electrical current from one electrode repels the molecules while the other electrode simultaneously attracts the molecules. The frictional force of the gel material acts as a "molecular sieve," separating the molecules by size.Their rate of migration through the electric field depends on the strength of the field, size and shape of the molecules, relative hydrophobicity of the samples, and on the ionic strength and temperature of the buffer in which the molecules are moving. After staining, the separated macromolecules in each lane can be seen in a series of bands spread from one end of the gel to the other. Applications Gel Electrophoresis is one of the staple tools in molecular biology and is of critical value in many aspects of genetic manipulation and study. One use is the identification of particular DNA molecules by the band patterns they yield in gel electrophoresis after being cut with various restriction enzymes. Viral DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified in this way. Another use is the isolation and purification of individual fragments containing interesting genes, which can be recovered from the gel with full biological activity. to determine the genetic difference and the evolutionary relationship among species of plants and animals. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid. The protein molecules in a sample of fish muscle tissue and plant grain endosperm tissue can be separated according to their individual molecular mass and compared to samples that have been treated with a reducing agent, such as 2-mercaptoethanol. Complex proteins (composed of two or more polypeptide chains) can be broken down into their respective polypeptide fractions. A reducing agent breaks the disulfide bonds that hold the polypeptide together. Electrophoresis of Nucleic Acids An agarose or polyacrylamide gel is loaded with the DNA fragments and current is passed through the gel. Since DNA is negatively charged, it will migrate towards the positive pole. The DNA will not migrate at the same rate, however. Larger pieces of DNA collide with the gel matrix more often and are slowed down, while smaller pieces of DNA move through more quickly.

10 Since different genes have different nucleotide sequences, restriction enzymes will cut them at different places, generating different size DNA fragments. By using gel electrophoresis, biologists can tell which gene is which based upon the sizes of the fragments generated when a gene is treated with a restriction enzyme. Agarose gel electrophoresis of DNA Most DNA molecules and their fragments are larger than proteins. Most DNA fragments are unable to enter a polyacrylamide gel, hence the larger pore size of agarose gel is required. Since the charge per unit length of DNA in any given fragment of DNA is the same ( due to the phosphate groups ) all DNA samples should move towards the anode with the same mobility under an applied electric field. The mobility of a DNA fragment depends on the size, the smallest molecules moving fast. Gel conc should be chosen to suit the size range of molecules to be separated. 0.3 % gels Separates DNA molecules between 5 and 60 Kb size. 2 % gels -- Separates DNA molecules between 0.1 and 3 kb size. 0.8 % gels Separates in the range of 0.5- 10 kb size. The M r of a DNA fragment can be determined by running a number of standard markers of known Mol. mass on the same gel. Bromophenol Blue is used as the tracking dye in the sample solvent. No stacking gel is needed. For viewing the gel, the fluorescent dye ethidium Bromide is used and then viewed under UV light. EB is a cyclic planar mol that binds between the stacked base pairs and fluoresce under UV light. Restriction endonuclease methods Restriction endonuclease is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are commonly classified into three types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially. These enzymes are routinely used for DNA modification in laboratories, and are a vital tool in molecular cloning. Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. The recognition sequences usually vary between 4 and 8 nucleotides, and many of them are palindromic, meaning the base sequence reads the same backwards and forwards. Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IV) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements,[32][33] as summarized below: Type I enzymes (EC 3.1.21.3) cleave at sites remote from recognition site; require both ATP and S-adenosyl-L-methionine to function; multifunctional protein with both restriction and methylase (EC 2.1.1.72) activities.

11 Type II enzymes (EC 3.1.21.4) cleave within or at short specific distances from recognition site; most require magnesium; single function (restriction) enzymes independent of methylase. Type III enzymes (EC 3.1.21.5) cleave at sites a short distance from recognition site; require ATP (but do not hydrolyse it); Sadenosyl-L-methionine stimulates reaction but is not required; exist as part of a complex with a modification methylase (EC 2.1.1.72). Type IV enzymes target modified DNA, e.g. methylated, hydroxymethylated and glucosylhydroxymethylated DNA Nucleic acid hybridization and the southern blotting Nucleic acid hybridization The bases in DNA will only pair in very specific ways: G with C and A with T In short DNA sequences, imprecise base pairing will not be tolerated Long sequences can tolerate some mispairing only if hydrogen bonding of the majority of bases in a sequence exceeds the energy required to overcome mispaired bases The source of any single strand of DNA is irrelevant, merely the sequence is important, thus complimentary DNA from different sources can form a double helix This phenomenon of base pairing of single stranded DNA strands to form a double helix is called hybridization as it may be used to make hybrid DNA composed of strands from different sources Because DNA sequences will seek out and hybridize with other sequences with which they base pair in a specific way much information can be gained about unknown DNA using single stranded DNA of known sequence Short sequences of single stranded DNA can be used as probes to detect the presence of their complimentary sequence in any number of applications including: Southern blots Northern blots (in which RNA is probed) In situ hybridization Dot blots . . . In addition, the renaturation, or hybridization, of DNA in solution can tell much about the nature of organisms genomes Library Screening The most common method of library screening involves hybridization of probes to target DNA Hybridization refers to the specific way DNA sequences base pair with their exact compliment Probes - Single stranded nucleic acids used to hybridize with a target DNA. Generally probes are radioactive or marked in some other way so that they can easily be identified after binding to target DNA To design probes for hybridization screening, something must be known in advance about the target sequence Hybridization Screening Takes advantage of the fact that complimentary strands of DNA can recognize one another By sticking DNA from many colonies or plaques in a library to a membrane Making the DNA single stranded Then hybridizing a probe to the DNA on the membrane thus marking target DNA on the membrane, colonies or plaques containing the target DNA can be identified

12 Southern Blots Called Southern blots after their inventor Involve four steps: 1 Digestion of DNA using restriction enzymes 2 Separation of the DNA fragments by size using gel electrophoresis 3 Transfer of fragments to a nitrocellulose or nylon membrane 4 Hybridization of a probe to the fragment or fragments of interest 5 Probe detection (autorad development) Hybridization labels DNA sequencing Real Time Polymerase Chain Reaction Minimal Residual disease Infectious disease load Current Developments