Section 8 Flashcards

Question

What is phosphodiester bonds?

Answer 1

- bonds that join nucleotides in nucleic acid - 5’ phosphate group of 1 sugar attaches to 3’ hydroxyl group of adjacent sugar - a molecule of H20 splits off

Answer 2

- function = carries genetic information. Serves as template for synthesis for RNA - location = nucleus (except mitochondrial DNA) - composition = repeating nucleotides linked by phosphodiester bonds between 5’ phosphate group of 1 sugar and 3 hydroxyl group of next - sugar = deoxyribose - pyrimidine = C, T - purines = A, G - structure = double stranded. Each strand has 5’ end (phosphate group attached to 5’C) and 3’ end (OH group attached to 3’C). Stranded are antiparallel. 2 strands coil around each other to form double helix with alternating sugar and P groups on exterior and bases on interior. Strands held together by hydrogen (H) bonds between complementary bases

Answer 3

- function = Converts genetic information from DNA into proteins. Some has regulatory of structural function - source of genetic information in RNA viruses - location = nucleus and cytoplasm - composition = repeating nucleotides linked by phosphodiester bonds between 5’ phosphate group of 1 sugar and 3’ hydroxyl group of next - sugar = ribose - pyrimidine = C, U - purines = A, G - structure = single stranded. Shorter than DNA. Irregular 3D structure

Answer 4

- phosphate 1 = adenosine monophosphate (AMP) - phosphate 2 = adenosine diphosphate (ADP) - phosphate 3= adenosine triphosphate (ATP)

Answer 5

- phosphate 1= Guanosine monophosphate (GMP) - phosphate 2= Guanosine diphosphate (GDP) - phosphate 3= Guanosine triphosphate (GTP)

Answer 6

- phosphate 1= cytidine monophosphate (CMP) - phosphate 2= cytidine diphosphate (CDP) - phosphate 3= cytidine triphosphate (CTP)

Answer 7

- phosphate 1= uridine monophosphate (UMP) - phosphate 2= uridine diphosphate (UDP) - phosphate 3= uridine triphosphate (UTP)

Answer 8

- phosphate 1= deoxyadenosine monophosphate (dAMP) - phosphate 2= deoxtyadenosine diphosphate (dADP) - phosphate 3= deoxyadenosine triphosphate (dATP)

Answer 9

- phosphate 1= deoxyguanosine monophosphate (dGMP) - phosphate 2= deoxyguansoine diphosphate (dGDP) - phosphate 3= deoxyguanosine triphosphate (dGTP)

Answer 10

- phosphate 1= Deoxycytidine monophosphate (dCMP) - phosphate 2= deoxycytidine diphosphate (dCDP) - phosphate 3= deoxycytidine triphosphate (cCTP)

Answer 11

- phosphate 1= deoxythymidine monophosphate (dTMP) - phosphate 2= dexoythymidine diphosphate (dTDP) - phosphate 3= deoxythymidine triphosphate (dTTP)

Answer 12

- reproduction of DNA during cell division

Answer 13

- newly synthesized strand of DNA by breaking bases hydrogen bonds to form 2 single-stranded templates is conserved

Answer 14

- enzyme that unwinds suction of DNA by breaking bases hydrogen bonds to form 2-single stranded templates for replication

Answer 15

- location where double stranded DNA (dsDNA) separates into single stranded DNA (ssDNA) and synthesis of DNA begins

Answer 16

- strand of DNA that serves as pattern for new strand of DNA or RNA

Answer 17

- RNA polymerase that synthesizes RNA primers needed to intiate replication

Answer 18

- segment of RNA formed on DNA template to start replication - provides 3’OH needed for phosphodiester bond information with incoming nucleotide

Answer 19

- enzyme that synthesizes daughter DNA by reading code on DNA template - can only synthesize DNA from 5’ end to 3’ end because 3’OH need to receive incoming nucleotides and form phosphodiester bonds - can only extend a nucleotide chains; can’t start one - needs primer to start

Answer 20

- synthesis of daughter strands of DNA from 3’-5’ target strand - DNA polymerase proceeds in linear fashion, assembling series of joined nucleotides in leading strand

Answer 21

- synthesis of daughter strand of DNA from 5’-3’ target strand - DNA polymerase must read target in 3’-5’ direction, so must proceed in nonlinear fashion, reading a section, assembling appropriate nucleotides, then moving back upstream to read another section in 3’-5’ direction. - new strand called lagging strand - consists of unconnected DNA fragments called Okazaki fragments separated by RNA primers

Answer 22

- daughter strand synthesized continuously from 3’-5’ template

Answer 23

- daughter strand synthesized discontinuously from 5’-3’ template

Answer 24

- short fragments of unconnected DNA formed during replication of 5’-3’ template

Answer 25

- enzyme that removes RNA primers from daughter strands of DNA

Answer 26

- enzyme that joins DNA fragments together

Answer 27

- helicase unwinds section of dsDNA to form replication forks - both original strands of DNA will serves as templates - replication will proceed simultaneously on both strands, in opposite directions

Answer 28

- primase reads section of code on DNA, attaches complementary nucleotides starting at 3’ OH end needed to grow nucleotide chain

Answer 29

- DNA polymerase reads template, attaches complementary nucleotides starting at 3’ OH of primer - code is read in 3’-5’ direction - synthesis on 3’-5’ templates is continuous, forming leading strand - synthesis on 5’-3’ template is discontinuous, forming lagging strand of disconnected Okazaki fragments

Answer 30

- RNase H removes RNA primers

Answer 31

- DNA polymerase places nucleotides where primers used to be

Answer 32

- DNA ligase joins DNA fragments together

Answer 33

- strand separation - synthesis of primers - extension of primers - removal of primers - replacement of primers - joining of Okazaki fragments

Answer 34

- synthesis off specific proteins based on DNA code

Answer 35

- RNA that transcribes DNA code in nucleus and carries it to cytoplasm where its translated into protein - about 1-2% of total cellular RNA

Answer 36

- RNA that reads code in mRNA and transport amino acids to growing polypeptide chain

Answer 37

- tiny regulatory RNAs involved in control of gene expression

Answer 38

- conversion of genetic information from DNA into complementary strand of mRNA - if DNA sequence is AGCT, mRNA sequence will be UCGA

Answer 39

- conversion of genetic information from mRNA into specific sequence of amino acids to form specific protein - takes place on ribosomes in cytoplasm - tRNA reads mRNA and assembles amino acids

Answer 40

- strand of DNA that doesn’t serve as template for synthesis of mRNA - its sequence is same as mRNA that is translated into protein (except it has T where RNA has U)

Answer 41

- strand of DNA that serves as template for synthesis of mRNA

Answer 42

- region of DNA that binds RNA polymerase, initiating transcription

Answer 43

- 3 nucleotides that code for an amino acid - 64 codons code for 21 amino acids - 2-6 codons for each, except methionine and tryptophan which only have 1

Answer 44

- AUG, codes for methionine

Answer 45

- UAA, UAG, and UGA. - not associated with any amino acids. - signal to stop protein synthesis

Answer 46

- 3-nucleotide sequence on tRNA that attaches to codon on mRNA, bringing with it specified amino acids

Answer 47

- organelles in cytoplasm and on surface of rough endoplasmic reticulum - made of rRNA and protein - site of proteins synthesis

Answer 48

- RNA polymerase binds to promotor region of gene - DNA separates into 2 single strands - RNA polymerase adds complementary nucleotides to produce strand of mRNA. if DNA sequence is 3’ ATTCGA 5’. MRNA will be 5’ UAAGCU 3’ - mRNA is released from DNA - DNA strands reanneal

Answer 49

- mRNA associates with ribosomes in cytoplasm - ribosome moves along mRNA, reading genetic code. Always begins with codon AUG - tRNA with complementary 3-nucleotide sequence (anticodon) attaches to codon to on mRNA, bringing with it amino acid specified by codon. E.g. anticodon UAC on tRNA attaches to codon AUG on mRNA, bringing with it amino acid methionine - as ribosomes moves along mRNA, other tRNAs bring in other amino acids - peptide bonds to form between amino acids - process continues until ribosome reaches stop codon - protein is released

Answer 50

- analysis of DNA and RNA; nucleic acid testing (NAT)

Answer 51

- isolation of DNA/RNA from other cellular components

Answer 52

- enzyme that degrades RNA - ubiquitous in environment

Answer 53

- enzyme that degrades DNA

Answer 54

- specific section of DNA under investigation

Answer 55

- pairing of complementary strands of nucleic acid, 1 from sample and 1 a reagent - resulting hybrids can be DNA:DNA, DNA:RNA or RNA:RNA

Answer 56

- product of hybridization

Answer 57

- stablility of bonding during hybridization - based on degreee of match and base composition - influenced by temp, pH, and salt concentration - increased temp, decrease salt ensure only most perfectly matched strands will remain paired

Answer 58

- short stand of DNA or RNA with known base sequence - isolated from organisms, cloned in bacteria, or synthesized - labeled with fluorescent or chemiluminescent dyes, enzymes, or radioisotopes to produce visible sign of hybridization, restriction fragment length polymorphism, conventional and real-time PCR - if complementary base sequence is present, probe, attaches to target

Answer 59

- matching d - used in reference to chromosome pairs

Answer 60

- EDTA - isolation of DNA and detection of viruses - 1 of preferred anticoagulants for blood and bone marrow

Answer 61

- K2EDTA and gel barrier - used for isolation of plasma - gel forms barrier between plasma and cells

Answer 62

- sodium citrate, gel, density, gradient fluid - used for isolation of mononuclear cells - gel forms barrier between mononuclear in plasma and RBCs/granulocytes

Answer 63

- acid citrate dextrose (ACD) - used for enhanced recovery of WBCs for several days after collection - 1 of preferred anticoagulants for blood and bone marrow

Answer 64

- heparin - generally not recommended - heparin inhibits polymerase; unacceptable for testing that involves PCR

Answer 65

- nucleated cells are source of DNA - some recent techniques can detect nonnuclear DNA in plasma - Nucleic acid of infectious agents often detected in plasma, sometimes in host DNA - hemolysis can interfere with amplificaton.

Answer 66

- DNA obtained from nucleated cells

Answer 67

- detection of chlamydia, cytomegalovirus (CMV), and BK virus (associated with rejection of transplanted kidney)

Answer 68

- detection of intestinal pathogens - analysis of stool DNA proposed for detection of colorectal cancer

Answer 69

- detection of infectious agents and cancer cells

Answer 70

- diagnosis of sickle cell anemia, Tay-Sachs disease, thalassemia, other chromosomal abnormalities and inherited disorders - usually grown in tissue culture to increase # of cells for DNA extraction

Answer 71

- good source of DNA, especially for genotyping bone marrow recipients and patients who have been recently transfused - avoid use of mouthwash with phenol or ethanol - use sterile Dacron or rayon swabs with plastic shafts - calcium alginate swabs and wooden shafts may contain substances hat inhibit PCR

Answer 72

- formalin-fixed, paraffin embedded tissue: DNA degraded top low molecular weight (MW) fragments - can usually be amplified but southern blot can’t be used (requires high MW DNA) - fresh tissue, snap-frozen tissue, or tissue frozen at -80C in optimal cutting temperature compound (OCT): yields higher MW DNA, OCT must be completely removed before DNA extraction

Answer 73

- forensic analysis

Answer 74

- whole blood can be stored at 2-25C for 24-48 hours after collection - separate WBCs by: — differential lysis in hypotonic buffer or water (RBCs lysed, WBCs precipitated by centrifugation) OR —density gradient centrifugation in Ficoll isolate mononuclear - WBCs can be stored at -70C for up to a year

Answer 75

- remove from RBCs promptly and hold at -20C or below

Answer 76

- fresh or frozen; grind, homogenize or mince - embedded: deparaffinize in xylene, rehydrate in decreased concentrations of ethanol

Answer 77

- break cell walls (enzymes, detergents, NaOH, boiling, or glass beads) - Enzyme treatment — less damage to chromosomal DNA - NaOH and boiling yield ssDNA - not suitable for restriction enzyme analysis - OK for amplification

Answer 78

- centrifugation of whole blood to obtain buffy coat, differential lysis with water or hypotonic solution to separate WBCs from RBCs, density gradient, centrifugation in Ficoll to obtain mononuclear cells (settle below plasma, about polys/RBCs)

Answer 79

- cells ruptured by mechanical or chemical methods

Answer 80

- liquid-phase extraction: use of solvents — mulitple manual steps — limited sample throughout phenol and chloroform are hazardous - solid-phase extraction: DNA absorption onto silica on membrane filters or magnetic particles — most commonly used method — easier — can be automated

Answer 81

- precipitation in ethanol or isopropanol in high salt concentration - resuspension in H20 or Tris- EDTA (TE) buffer - handle with care to avoid breaking DNA

Answer 82

- extracted DNA can be stored at RT for several months, refrigerated for 1 year, or frozen at-20C to -70C for up to 10 years

Answer 83

- similar - lysis of cells, isolation by phenol-choloform extraction or binding to silica, precipitation in alcohol

Answer 84

- RNA less stable - process specimens promptly - can be stored suspended in ethanol for several months at -20C or long term at -70C

Answer 85

- RNA more susceptible to degradation - use RNase decontamination solution to clean gloves, benchtops, equipment; RNase-free reagents and plastic ware; nucleated-free water; DNase I to digest DNA

Answer 86

- technique to increased (amplify) amount of nucleic acid in sample, probe, or signal so that very small amounts of nucleic acid can be detected

Answer 87

Technique to increase amount nucleic acid in sample through in vitro replication, e.g. PCR, transcription mediated amplification (TMA)

Answer 88

- technique to increase amount of probe bound to target to so very small amounts of nucleic acid can be detected e.g. ligase chain reaction

Answer 89

- technique to increase signal generated so that very small amounts of nucleic acid can be detected, e.g. branched chain signal amplification (bDNA), e.g. hybrid capture assay (HCA)

Answer 90

- term used to describe tests that detect pathogens by presence of nucleic acids rather than antibodies - provides earlier detection. - frequently used for viral detection and in microbiology for bacteria

Answer 91

- 1st AMD most widely used amplification method - specific primers and thermos table DNA polymerase makes copies of target DNA by repeated cycles of denaturation, annealing and extension - can produce a million copies in less than an hour

Answer 92

- use of heat to separate strands of DNA

Answer 93

- in real-time PCR, Ct equals the number of cycles needed to produce a fluorescent signal that exceeds the background fluorescence - with some viruses, the Ct value can inversely related to viral load

Answer 94

- separation of ds DNA into 2 strands using heat or alkali

Answer 95

- temp required to denature 50% of dsDNA - depends in part on GC content - G-C pair harder to break than A-T pair because 3 H-bonds instead of 2

Answer 96

- binding to complementary strand of nucleic acid

Answer 97

- enzyme that assembles nucleotides to produce new strand of nucleic acid

Answer 98

- enzyme that synthesizes DNA using existing strand of DNA as template - can only ass to existing piece of DNA so needs primer to start

Answer 99

- heat-stable DNA polymerase used to catalyze synthesis of DNA in PCR - originally isolated from bacterium Thermus aquatics in hot springs of Yellowstone National Park - can withstand high temp required for denaturation

Answer 100

- short segements of DNA designed to anneal to DNA target in PCR - provide free 3’-OH group to which DNA polymerase can add dNTP

Answer 101

- short fragments of ssDNA that are synthesized for specific purpose

Answer 102

- building blocks from which DNA polymerase synthesizes new strands of DNA, dATP, dGTP, dCTP

Answer 103

- instrument that rapidly changes temp during amplification cycle

Answer 104

- product of amplification

Answer 105

- PCR amplicons are slowly heated in presence of dyes specific for dsDNA - at low temp, signals is high - as temp increase, DNA strands separate and signal decrease - fluorescent signal patterns vary depending on nucelotide sequence

Answer 106

- enzyme used in vitro to make DNA from RNA - isolated from retroviruses

Answer 107

- DNA copied from RNA by reverse transcriptase

Answer 108

- to join together by chemical process

Answer 109

- enzyme that links DNA strands by joining 5’ end of one to 3’ end of another

Answer 110

- DNA region to be amplified - from patients genomic or mitochondrial DNA or from microorganisms

Answer 111

- shot segments of ssDNA designed to hybridize to template strands and provide starting point for synthesis of daughter strand - 1 designed to hybridize adjacent to region of interest on 3’ end of 1 strand, other designed to hybridize adjacent to region of interest on 3’ end of other strand - segment to ab amplified falls between 2 primers

Answer 112

- building blocks form which DNA polymerase synthesizes a new stand of DNA: dATP, dGTP, dTTP, dCTP

Answer 113

- e.g. Tag polymerase - extends primers on each template strand of dNTPs and catalyzing formation of phosphodiester bonds

Answer 114

- denaturation (melting) - Annealing (hybridization) - extension (polymerzation)

Answer 115

- “melting” - temp = 90-96C/20-60 seconds - dsDNA separated into 2 strands by heat - each strand serves as template for synthesis of new strand

Answer 116

- “hybridization” - temp = 50-70C/20-90 seconds - primers attach to both template strands by binding with complementary bases on 3’ side of region to be amplified

Answer 117

- “polymerization” - temp = 68-75C/ 10-60 seconds - DNA polymerase attaches to ends of primers, adds dNTPs that are complementary to template, synthesizing new strands of DNA

Answer 118

- repeated 15-40 times - each cycle results in doubling of DNA, yielding millions of copies of original DNA segment - amplicon can be analyzed by variety of methods

Answer 119

- contains nucleic acid sequence of interest - purpose = ensures that polymerase is active, primers and thermocycler working properly - expected reaction = target sequence detected

Answer 120

- contamination control, reagent blank, or no target control) - contains all PCR components except DNA - purpose = checks for contamination with target DNA or amplicon from previous amplification - expected reaction = target sequence not detected

Answer 121

- contains DNA but not target DNA - purpose = ensures that primers aren’t binding to wrong DNA sequences - expected results = target sequence not detected

Answer 122

- amplification control - contains 2nd set of primers and unrelated target (housekeeping gene or other nucleic acid target introduced in sample before nucleic acid extraction or during (PCR) - purpose = demonstrates that PCR is working. Differentiates true NEG from false NEG due to amplification failure. Detects PCR inhibitors - expected reaction = housekeeping gene detected

Answer 123

- uses 2 pairs of primers and second round of amplification to increase sensitivity and specificity

Answer 124

- simultaneous amplification and detection (fluorescence) in sealed tube - less subject to contamination - eliminates need to analysis of product by gel electrophoresis - quantitative by analyzing the Ct - commonly used for detection of microorganisms

Answer 125

- uses more than 1 set of primers so mulitple targets can be amplified in same tube

Answer 126

- method to amplify RNA (mRNA or microbial RNA) - same procedure as PCR except RNA is 1st converted to cDNA by reverse transcriptase - used to detect viruses such as SARS-Cov-2, HIV and Hepatitis C virus (HCV), measure gene expression

Answer 127

- type of transcription-based amplification systems (TAS) - non-PCR methods to amplify target RNA - similar methods, different manufacturers - cDNA is formed from target RNA - millions of copies of RNA produced by transcription of cDNA - assays to detect RNA viruses, mycobacterium tuberculosis, chlamydia trachomata’s - TMA and NASBA are isothermal run - don’t require thermocycler

Answer 128

- type of probe amplification - pairs of probes, each consists of 2 unconnected oligos that bind adjacent to the template - 1 segment is fluorescently labeled - if both bind, DNA ligase joins them and DNA polymerase copies the probe - applications = Duchenne muscular dystrophy

Answer 129

- type of signal amplification - probes capture target sequence to SLE support - extender, preamplifier, and amplifier probes hybridize sequentially - final probe is branched and carries signal-generating enzymes that act on chemiluminescent substrate - application = detection of HBV, HCV, CMV; HIV viral loads

Answer 130

- type of signal amplification - complexes containing target DNA bind to antibodies on solid support - detected by enzyme-labeled antibodies - application = detection of HPV

Answer 131

- isothermal method that uses primary probe, invader probe reporter probe - enzyme cleavage amplifies reporter probe bound to DNA target - application = detection of cystic fibrosis, factor Leiden, HPV

Answer 132

- dot/slot blot - sandwich hybridization - line probe assay

Answer 133

- sample applied to membrane - membrane heated to denature DNA and labeled probe added - unhybridized probe washed away - hybridized probe visualized by autoradiography or enzyme assay - POS rxn indicates presence of target sequence - application = detection of mutations associated with genetic diseases such as sickle cell anemia

Answer 134

- unlabeled probe bound to membrane or well surface (capture probe) - sample applied. If target sequence is present, anneals to capture probe - labeled probe (signal-generating probe) that anneals to different site on target added - more specific than dot blot - can be carried out in microtiter plates plates, so adaptable to automation

Answer 135

- probes attached to strips, amplified target added - unbound target washed away - hybridized complexes visualized with Biotin-streptavidin method - application = infectious diseases

Answer 136

- target nucleic acid and probe interact in aqueous solution. - rapid and more sensitive than solid phase - uses smaller sample size. - adaptable to automation - application = detection of bacterial rRNA in clinical specimens or cultures

Answer 137

- target nucleic acid detected in intact cells - tissue or cells fixed on glass slide - Probe added. Attached to complementary DNA sequences - application = cytogenetics studies

Answer 138

- hybridization is performed directly on intact chromosomes using a fluorescent-labeled probe - fluorescent microscope used with appropriate filters to transmit the excited light from the specific probes - application - IgH gene translocation such as t(11:14) in multiple myeloma, BDR/ABL (for CML or ALL) aneuploidy and chromosomal deletions

Answer 139

- mulitple probes immobilized as specific locations on solid support (glass slide, silicone chip) - after amplification, sample and control nucleic acid labeled with 2 different fluorescent dyes and loaded onto chip - sample and control compete to hybridize to chip based on relative amounts - unbound strands of target washed away - hybridized nucleic acid detected with fluorescent detector - relative colors measured - thousands of reactions can be performed at same time - application = gene profiles associated with certain cancers/diseases, classification of leukemias, tumor staging, determination of drug resistance in HIV

Answer 140

- nucleic acids migrate to POS pole (anode) in electrical field due to NEG charge on phosphate group - molecules with smallest MW travel furthest from point of application. - traditional method to detect/analyze nucleic acids following isolation - matrices = agarose, polyacrylamide - buffer = tris-acetate-EDTA (TAE), tris-borate-EDTA (TBE) - tracking dyes = bromphenol blue, xylene cyanol - stains = ethidium bromide (most commonly used fluorescent DNA-binding dye; mutagen, handle with care), SYBR gold, picoGreen and YOYO-1 - applications = PCR/restriction fragment length polymorphism (RFLP), RT-PCR fragment analysis, Southern and Northern blotting, DNA sequencing, serum protein and CSF analysis

Answer 141

- polycrylamide gel electrophoresis (PAGE) - capillary electrophoresis - pulse-field gel electrophoresis - denaturing gel electrophoresis

Answer 142

- used when DNA fragments are small - high degree of resolution - can differentiate fragments that differ by just 1 base pair

Answer 143

- performed in thin capillary tubes using high voltage - utilizes electroendrosmosis, which draw molecules towards the cathode where in UV light or fluorescence detector molecules as they pass by. - used in DNA sequencing and fragment analysis

Answer 144

- alternates direction of current - used in epidemiology of infectious diseases

Answer 145

- uses chemicals to denature secondary structures - used most often in electrophoresis of RNA

Answer 146

- nucleic acids that have been separated by electrophoresis are transferred to solid support membrane

Answer 147

- used to analyze DNA containing a specific base sequence by applying a probe to DNA bands immobilized onto nitrocellulose paper after electrophoresis - useful for ID of DNA fragments that don’t amplify well, for polymorphisms and structural alterations

Answer 148

- determination of order of nucleotides in DNA molecule - used to detect mutations, type microoraganims, establish treatment for HIV and HCV

Answer 149

- Sanger method - widely used method to determine DNA sequence

Answer 150

- ddATP, ddGTP, ddCTP or ddTTP - chain terminating nucleotides used in DNA sequencing - lack of 3’-OH group required for formation of phosphodiester bonds between nucleotides - when in corporates into growing DNA strand, extension stops.

Answer 151

- 4-lane gel electrophoresis pattern obtained from dideoxy chain termination sequencing

Answer 152

- most commonly used method for DNA sequencing in clinical lab - based on Sanger method. - Amplification by PCR, denaturation, annealing or primer, extension of new strand by DNA polymerase - Extension stops when ddNTP is incorporated. - ddNTPs are labeled with different fluorescent tags, detected by capillary electrophoresis and fluorescent detector

Answer 153

- method to determine DNA sequence in short segments based on generation of light - doesn’t recruiter electrophoresis or sequencing ladder

Answer 154

- permanent change in nucleotide sequence - may of may not cause disease

Answer 155

- mutation involving single nucleotide pair

Answer 156

- variation in DNA sequence that occurs in at least 1% of population - some cause susceptibility to disease, or affect the response to drugs

Answer 157

- “snip” - variation in single base - most common type of variation in human genome - example of disease caused of hereditary hemochromatosis (single base mutation in HFE gene on chromosome 6

Answer 158

- bacterial enzymes used to cut DNA at specific sites - used to produce short fragments for electrophoresis

Answer 159

- nucleotide sequence recognized by restriction endonuclease - most are palindromes

Answer 160

- nucleotide sequence that reads same in both directions e.g. the complementary strand to 5’-CATG is 3’-CATG - since reading is from 5’ to 3’ direction, both are read as CATG

Answer 161

- DNA fragments formed by action of restriction endonuclease on DNA

Answer 162

- Pattern of DNA fragments produced following digestion with restriction endnucocleases - fragments separated by gel electrophoresis - stained with ethidium bromide and viewed under UV light or blotted onto nitrocellulose membrane and detected with labeled probe (Southern Blot)

Answer 163

- differences in restriction patterns due to variations in nucleotide sequences that change where restriction enzymes cleave DNA - useful in forensics and paternity testing, detection of genes associated with specific diseases, ID of microorganisms

Answer 164

- modification of DNA replication that incorporates labeled dideoxynucleotides (ddNTPs, chain terminating nucleotides) in reaction mixture - similar to sNTPs except lack 3’OH needed for phosphodiester bond formation - when incorporated into growing DNA chain, elongation is terminated - components = template, primer, DNA polymerase, dNTPs, ddNTPs

Answer 165

1. DNA template amplified 2. 4 reaction tubes containing” — template — primer — DNA polymerase — all 4 dNTPs — only 1 ddNTP 3. Amplicon denatured 4. Primer hybridizes to target 5. DNA polymerase extends primer, occasionally incorporating ddNTP that stops further extension. Resulting fragments are of various lengths. All fragments in tube end with same labeled ddNTP 6. Product electrophoreses in 4 separate lanes labeled A, C, G, T, corresponding with the ddNTP in tube. Fragments separate according to size 7. Gel dried and exposed to x-ray film, producing sequencing ladder. Band furthest from origins is smallest, fastest migrating fragment and ends in the 1st nucleotide in the sequence, e.g. if band futhest from origins is in lane A (lane from tube that contained ddATP), then 1st nucleotide in sequence is A. Ladder is read from bottom to top to determine entire nucleotide sequence

Answer 166

- uses fluorescent labels and capillary gel electrophoresis - fragments migrate through gel according to size, pass by laser beam and detector - bases identified by color of fluorescence - results in electropherogram rather than gel pattern - application = detection of mutations, genotyping HIV to determine drug resistance and HCV to determine therapy and prognosis

Answer 167

- principle = parallel sequencing of small pieces (subunits) of DNA where each subunit is sequenced rapidly numerous times followed by assembling the fragments to yield a sequence of the starting material - NGS allows rapid sequence with a much smaller amount of DNA when compared to the Sanger methodology - components = sample, primer, DNA polymerase, dNTPs, reference genome - application = detection of mutations for a variety of diseases, detect target cancer drug resistance mutations, detection of cell-free tumor DNA (liquid biopsy)

Answer 168

1. Library preparation (prepares DNA or RNA to be comparable with a sequencer) — sonication 2. Cluster generation — clones produced of each subunit — separation of clones in wells, on beads or on a glass slide 3. Sequencing (bridge amplification) — complementary strands made to amplicon of each single clone 4. Data analysis — instrument software and external programs can aid in this process

Answer 169

- High sensitivity - high specificity - ability to predict one disease before they are manifested in patient - ability to detect mutations that predict response to some therapies - ability to detect nonviable, fastidious slow-growing and nonculturable microorganisms - adaptability to automation (high throughput, rapid turnaround time)

Answer 170

- potential for nucleic acid contamination - Data not always clinically significant e.g. presence of microbial nucleic acid doesn’t indicate disease, unable to differentiate viable and dead microorganisms - cost

Answer 171

- contamination from other specimens - contamination with amplicons from previous target amplification - environmental contamination - contamination by testing personnel

Answer 172

- use of plugged pipette tips and screw cap tubes to minimize sample aerosols

Answer 173

- use of closed tube (real-time) PCR or signal or probe amplification methods; separate areas for sample prep ad amplification with no movement of equipment or reagents from amplification area to sample prep area; use of dUTP-UNG system to destroy amplicons form previous amplifications

Answer 174

- use of 10% bleach or alcohol to clean benches, hoods; UV light to decontaminate sample prep areas (interferes with DNA replication by breaking sugar-phosphate backbone)

Answer 175

- use of lab coats, and gloves

Answer 176

- inhibitors in sample - degradation of nucleic acids during transport/handling - degradation of nucleic acid during processing - degradation of nucleic acid during storage

Answer 177

- proper specimen collection (correct anticoagulant, no hemolysis), use of internal control or split sample testing (1 aliquot with target added)

Answer 178

- use of transport media, collection tubes designed to stabilize RNA, prompt processing or preservation, inhibiton of DNases and RNases

Answer 179

- use of glover when handling samples, standards, controls to protect against nuclease degradation - RNase-free conditions for isolation of RNA

Answer 180

- storage at proper temp: — specimens: snap freeze and store at -70C — DNA: RT a few month or 1 year in refrigerator, -20C to -70C for 10 year — RNA: -20C few months, -70C longer - avoid repeated freeze/thaw cycles and frost-free refrigerators - can damage DNA

Answer 181

- detection of microorganisms - viral load testing - genotyping to determine antimicrobial resistance - epidemiology - classification (and reclassification)

Answer 182

- viral load testing - measurements of cytokine activity

Answer 183

- detection of point mutations (using Sanger or NGS method)

Answer 184

- diagnosis and classification of B and T cell leukemias and non-Hodgkins Lymphomas - monitoring of therapy and remission

Answer 185

- screening donors for bloodborne pathogens - fetal typing for risk of HDFN - determining Rh variants - determining blood group variants - antigen status of chronically transfused patients

Answer 186

- diagnosis of genetic disease - diagnosis of carrier state

Answer 187

- HLA typing (PCR based) - detection of polymorphisms in major histocompatibility complex MHC genes (NGS often utilized)

Answer 188

- diagnosis of cancer - mutations to predict lack of or favorable response to therapies

Answer 189

- selection of optimal drug therapy based on patients genotype - ID of potential adverse drug affects based on patients genotype

Section 8 Flashcards

(213 cards)