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Molecul. Diag. Tech
Molecular Diagnostic Techniques
| Question | Answer |
|---|---|
| Are made up of nucleotides that occur in very specific sequences in different cells and microorganisms. | DNA and RNA |
| ________ is a double-stranded molecule w/ very specific base pair linkages: Adenine-Thymine, guanine-cytosine | DNA |
| When the molecule is replicated, the styrends are _____, and a new strand is created that is complementary to the parent strand. | separated |
| The exact base pairing foms the baeses for identification of unknown sequences through the use of known short nucleic acid sequences called _________ | probes |
| The pairing of a probe w/ a complementary DNA strand is called________. Probe is labeled w/ a marker for detection. | hybridization |
| Dot-blots and sendwich hybridization techniques are used to directly identify specific ________ in patient sample. | sequences (probes) |
| Detailed characteristics of the DNA is done by the use of enzyme _________ that cleave DNA at specific recognition sites. | restriction endonucleases |
| Specdific microrganisms can be identified on the basis of unique paterns obtained, called ______ | RFLPs |
| when intact cells or tissues is used ____ takes place | in situ hybridization |
| It is used to identify alterations of genes in various cancers | chip technology |
| The most widely used method of target amplification | PCR polymerase chain reaction |
| Probes ruther than target nucleic acid can be amplified by _________ | LCR ligase chain reaction |
| All amplification techniques are subject to ________ reactions resulting from eighter contamination with previously amplified products or background reactivity. | false-positive |
| Technology that generates multiple copies of a specific target nucleotide sequence from a target organism | PCR polymerase chain reaction |
| The genetic sequence refers to the order of the ________(bases) | nucleotides |
| Ex: sequence is CATG. It will only bond (hybridize) with its complement ____ | GTAC |
| Specific sequence of base pairs codes for specific ______ representative of each organism | amino acids |
| _______ contains highly conserved coded regions common for the organism; (Variable regions resulting in individual ________. | Genome; traits_ |
| Selection of a highly conserved area enables high_________ for PCR method | specificity |
| Formation of a linear amino acid chain using the mRNA strand is called________ | Translation |
| Normally a very stable molecule | DNA |
| Heat will cause denaturing rezulting in __________ | Strands separation |
| Cooling results in ____ rezulting in complementary strands to rejoin | annealing |
| In molecular testing the ______ is mannipulated. | replication of DNA |
| Procedure is done by : extraction, amplification and ________ | detection |
| Extraction ia done from the ______ | the specimen |
| Steps to ______________ procedure: Separate DNA strands; Target specific sequence; Attach primers & add polymerase; Replicate target sequence & repeat. | Amplification |
| Used for Detection in molecular testing | enzyme, fluorescent or chemical means |
| Used in moleculat testing: Test DNA sample; _________ primers; ________ DNA polymerase (Taq); Nucleotide triphosphates (ATP, GTP, CTP, TTP) | Oligonucleotide; Thermostable |
| It is done: Usually >90 C separates double stranded DNA into two single strands; The ________ holding the bases together are weak, unlike the bonds between the deoxyribose & phosphates | Denaturation by Heat; hydrogen bonds |
| Step 2: Annealing-Primer Binding to the Target means not to replicate the entire strand but just the target sequence; often ______ base pairs | 100-600 |
| Always begins at 3’ end of the primer, making double strands of the single strands | Extension |
| When Two new DNA strands identical to the original target form, the ________ is done. | End of the first cycle |
| Molecular Diagnostic techniques are used to: Detection of Hgb ________; Numerous viral and microbial organisms, Human Immunodeficiency virus, Hepatitis C.Human PapillomavirusesWest Nile Virus | S variant |
| _______ carries the primary genetic information within chromosomes found in each cell | DNA |
| _____ is an intermediate nucleic acid structure that helps convert the genetic information encoded within DNA into proteins. | RNA |
| Both nucleic acid structures are ______ made up of repeating nucleotides | polymers |
| The sugar deoxyribose is present in ________, while ribose is the primary sugar found in ____. | DNA; RNA |
| DNA and RNA have the same two _____ bases, adenine and guanine, but the _____ bases differ. | purine; pyrimidine |
| DNA uses cytosine and thymine; RNA uses cytosine and _______. | uracil |
| DNA exists primarily as a ____ molecule with very specific base pair linkages: adenine pairs with thymine, and cytosine pairs with ______. | double-stranded; guanine |
| The two strands are twisted into an ___________ | alpha helix. |
| Hydrogen bonding between adjacent bases holds the ______ together | two chains |
| ______is characterized as a semi-conservative process, because one strand of the molecule acts as a template for creation of a complementary strand. | Replication of DNA |
| When DNA replicates, ________molecules result, each of which is an exact copy of the original molecule. | Two daughter |
| The double helix can easily be separated or denatured using heat or an _______ solution | alkaline |
| When heat is used to separate the strands, the process is known as ________ | melting |
| __________ is the generation of a strand of messenger RNA (mRNA) that codes for the gene that is to be expressed as a protein. | Transcription |
| _______ occurs when the mRNA travels from the nucleus into the ________ of the cell to ribosomes to guide protein synthesis. | Translation; cytoplasm |
| All forms of RNA are _______polymers with an irregular _________ structure of much shorter lengths than DNA | single-stranded; three-dimensional |
| ____________ is used to translate the DNA code into making functional proteins. | Messenger RNA (mRNA) |
| _______ transports amino acids to make proteins. | Transfer RNA (tRNA) |
| _________ acts as the site for protein synthesis directed by the mRNA. | Ribosomal RNA (rRNA) |
| ___________ is a short strand of DNA or RNA of a known sequence that is used to identify the presence of its complementary DNA or RNA in a patient specimen. | A nucleic acid probe |
| The two strands should share at least ____ consecutive bases of perfect _______ to form a stable hybrid. | 16 to 20 ; complementarity |
| Probes are labeled with a ______, such as a radioisotope, a fluorochrome, an enzyme, or a chemiluminescent substrate to make detection of hybridization possible. | marker |
| ________ can take place either in a solid support medium or in solution. | Hybridization |
| ___________ assays are the simplest types of solid support hybridization assays. | Dot-blot and sandwich hybridization |
| In the dot-blot assay, clinical samples are applied directly to a ___________. | membrane surface |
| A positive result indicates presence of a specific __________. | sequence of interest. |
| ___________ uses two probes, one of which is bound to the membrane and serves to capture target sample DNA; The second probe anneals to a different site on the target DNA, and it has a label for detection. | Sandwich hybridization |
| Sandwich hybridization assays have been developed using ___________ plates instead of membranes, which is more adaptable to automation. | microtiter |
| To characterize DNA present in a patient sample in a more detailed fashion, enzymes called ___________ are often used. | restriction endonucleases |
| _______ cleave both strands of a double-stranded DNA at specific recognition sites, yielding fragments that are approximately 4–6 base pairs long. | enzymes |
| The resulting fragments are separated from each other on the basis of molecular weight and charge by gel __________. | electrophoresis |
| The gel can either be stained with ________ and viewed under ultraviolet light, or specific nucleic acid sequences can be identified through the use of DNA probes | ethidium bromide |
| Differences in restriction patterns are referred to as restriction fragment length polymorphisms | RFLPs |
| Used to detect polymorphisms in major histocompatibility complex (MHC) genes, or to obtain a DNA fingerprint of a particular individual. | RFLPs |
| Analysis of DNA fragments using probes is typically carried out using a technique known as a _______ assay, or Southern blot | Southern hybridization |
| After DNA fragments are separated by gel electrophoresis, the fragments are denatured using _______. | alkali |
| Southern blots are useful to analyze alterations of ________ of DNA, where amplification by the polymerase chain reaction (PCR) may not be practical. | large spans |
| Southern blot analysis can reveal __________ in DNA sequence based on the RFLP profile made visible by the | polymorphisms |
| Northern blots are performed in a similar manner to study ___. Used most often to determine the level of expression of a particular ________ RNA. | RNA; messenger |
| Hybridization assays can also be performed in a _________ phase | solution |
| both the target nucleic acid and the probe are free to interact in a reaction mixture; it happens during ___ | solution phase |
| __________ assays are fairly adaptable to automation, especially those using chemiluminescent labels. | Solution-phase hybridization |
| ___________ represents a third type of hybridization reaction in which the target nucleic acid is found in intact cells. | In situ hybridization |
| Formalin-fixed and paraffin-embedded tissue sections are typically used for _________ procedure | In situ hybridization |
| In situ hybridization technique is the same as immunohistochemistry except that _________ instead of antibodies are used as probes. | nucleic acids |
| This technique is used to detect a number of malignancies linked to chromosomal abnormalities. | fluorescent in situ hybridization (FISH). |
| _______, also called microarrays, are very small devices used to examine DNA, RNA, and other substances. | Biochips |
| Hybridization occurs on the chip’s surface, allowing thousands of hybridization reactions to occur at one time. | Biochips |
| The intensity of the fluorescent signal at a particular location is proportional to the sequence ________ at a particular ______. | homology; locus |
| Since most ________(SNPs) are silent and have no apparent functional consequence, particular SNPs must be linked to particular disease entities. | single-nucleotide polymorphisms |
| Detection of point mutations by ________can be used for classifying leukemias, molecular staging of tumors, and characterizing microbial agents. | microarray |
| _________is considered the “gold standard” for many molecular applications | DNA sequencing |
| The sequencing reaction itself is based on the __________ reaction developed by Sanger and colleagues in 1977 | dideoxy chain termination |
| Target amplification systems are in _____ methods for the ______ replication of a target molecule to levels at which it can be readily detected. | vitro; enzymatic |
| polymerase chain reaction (PCR), transcription mediated amplification (TMA), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA) are ______ | Target amplification systems |
| ____requires several components for the reaction to occur: (1) a thermostable DNA polymerase , (2) deoxynucleotides of each base (3) the DNA of interest containing the target sequence, and (4) oligonucleotide primers. | PCR |
| _______ primers are short segments of DNA (20–30 nucleotides long), that are complementary to opposite strands. | Oligonucleotide |
| Each_______ consists of three steps: denaturing, annealing, and extending at different ____________. | PCR cycle; temperatures |
| During _________, the double-stranded DNA is heated to 95ºC to separate or denature the DNA into single strands. | denaturation |
| The mixture is then cooled to 52ºC, at which time the primers bind to, or _____ to, the complementary sequences on the separate DNA strands. | anneal |
| The third step, _________, takes place typically at 72ºC. | elongation |
| The resulting DNA fragments, referred to as __________, are detected using various methods. | amplicons |
| amplicons are detected using __________ probes. | labeled nucleic acid |
| The enzyme ____________ is used to generate copy DNA (cDNA) using the RNA as a template. Process known as ___________ | reverse transcriptase; reverse transcriptase PCR |
| One of the major uses for PCR is in testing for the presence of _____. | HIV |
| ______is also considered the gold standard for detecting __________ virus and monitoring therapy in those infected; identifying Mycobacterium tuberculosis and diagnosing early initial infection with (CMV). | RT-PCR;hepatitis C |
| The __________ system (TAS) was the first non-PCR nucleic acid amplification method that amplified an RNA target. | transcription-based amplification |
| nucleic acid sequence-based amplification (NASBA) and transcription mediated amplification (TMA); do not require the use of a thermal cycler. | non-PCR target amplification methods |
| _____(NASBA) uses the enzymes reverse transcriptase, RNase H, and bacteriophage T7 DNA-dependent RNA polymerase to isothermally amplify an RNA target | Nucleic acid sequence-based amplification |
| ______ (TMA) uses only two enzymes to drive the reaction: an RNA polymerase and a reverse transcriptase; isothermal like NASBA | Transcription-mediated amplification |
Created by:
tatianat