Term
| how do we get a good quantity and quality of DNA/RNA sample? (2) |
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Definition
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Term
| how can large quantities of DNA be isolated |
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Definition
| by transferring a vector of that DNA into a single-celled host and growing the microorganism in culture (cloning) |
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Term
| common bacterial and yeast hosts |
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Definition
| b - E. coli, y - S. cerevisiae |
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Term
| vector / what does it contain (3) |
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Definition
| DNA molecule that can replicate autonomously in a host / DNA sequence of interest, origin, one or more antibiotic resistance genes |
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Term
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Definition
| plasmids, bacteriophage lambda, cosmids, BACs, YACs |
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Term
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Definition
| bacterial artificial chromosome - very large fragments of dna |
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Term
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Definition
| yeast articial chromosome - largest capacity for dna |
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Term
| constructing a vector containing the DNA of interest requires use of restriction enzymes, also called |
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Definition
| restriction endonucleases - recognize specific double-stranded sequences in DNA and cleave by introducing a nick with a sticky end at each strand |
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Term
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Definition
| restriction enzyme, recognizes 5'-GAATTC-3' |
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Term
| plasmid and Dna are both _ by the same restriction enzyme. _ (enzyme) is used to create the new recombinant molecule |
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Definition
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Term
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Definition
| collection of recombinant clones from a source known to contain the gene, cDNA, or other DNA sequence of interest |
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Term
| localization and cloning of specific genes (4) |
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Definition
| linkage analysis to identify closely linked DNA markers, identifying the corresponding DNA clones such as YACs and BACs, and searching for the genes present in the clones. Does NOT require knowledge of function of the genes. |
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Term
| 2 approaches to molecular genetic testing at the DNA level |
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Definition
| direct - assay the genes of interest by specifically isolating them. indirect - look for markers known to be associated with the observed phenotype, even though specific genes may not be known |
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Term
| direct approach to molecular genetic testing at the dna level |
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Definition
| 1. gene of interest is cloned (need to know it) 2. disease causing mutations in the gene are characterized 3. diagnosis. (no family members needed, just the patient) |
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Term
| most common techniques for direct testing of dna (6) |
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Definition
| PCR, southern blot, allele specific oligonucleotides, microarray (chip) technology, protein truncation test, single strand conformation polymorphisms |
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Term
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Definition
| alternative to cloning. can amplify a fragment of DNA that is located between 2 oligonucleotide "primers" |
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Term
| PCR applied to the analysis of RNA is referred to _ because _ |
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Definition
| reverse transcriptase PCR / it is first copied into a cDNA library |
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Term
| PCR "exponential DNA synthesis" results from (3) (amplification?) |
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Definition
| repeated cycles of heat denaturation, hybridization of the primers, and enzymatic DNA synthesis of target sequence |
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Term
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Definition
| hair root, buccal swab, single blastomere (preimplantation diagnosis), forensics: sperm in a vaginal swab from a rape victim, drop of blood at a crime scene |
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Term
| southern blot technique overview |
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Definition
| direct, specific DNA sequence. involves transfer of DHA fragments - that have been electrophoretically separated - to a filter membrane, and subsequent detection of the fragment by probe hybridization |
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Term
| southern blot technique in more detail |
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Definition
| isolate genomic dna, restriction enzyme digestion to fragments (separated by size via agarose gel electrophoresis), denaturation into separate dna strands, which are transferred from the agarose gel to a filter paper by blotting. a labeled probe of ssDNA that is complementary to the sequence of interest is applied to the filter paper |
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Term
| SNoW DRoP / how do northern and western work |
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Definition
| southern - dna, northern - rna, western - protein. all work the same way as southern |
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Term
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Definition
| allele specific oligonucleotide test can be used for detection of point mutations. short oligonucleotides corresponding to normal and mutant sequences at a particular location are synthesized. normal control DNA and patient's DNA are amplified using PCR primers flanking the site of a potential mutation. PCR products are spotted on to filters in duplicate and hybridized to normal and mutant oligo probes separately. diagnosis made based on hybridization patterns |
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Term
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Definition
| (direct testing) amount and pattern of hybridization from a very large number of DNA fragments on a piece of silicon |
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Term
| DNA testing: indirect approach is used when |
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Definition
| the gene is not cloned but the location of the gene is known. linkage analysis is used. other family member's DNA is required. |
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Term
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Definition
| DNA markers close to disease locus are used to track the inheritance of the chromosome that harbors a mutant gene in a family. accuracy of the test results depend on distance between marker and locus. |
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Term
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Definition
| located close together, are inherited together (very low chance of recombination) |
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Term
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Definition
| recombination frequency of 1% = a million base pairs |
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Term
| indirect testing: overall technique (4) |
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Definition
| distinguish the two chromosomes of the parent; determine which marker allele at the chosen polymorphism segregates with the disease allele; determine which allele at the marker locus was inherited by the individual at risk; know the distance between the marker and the disease locus |
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Term
| dna markers used in linkage analysis (3) |
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Definition
| SNPs, microsatellite repeats, restriction fragment length polymorphisms (RFLPs) |
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Term
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Definition
| if a normal variation is present in a recognition sequence for a particular restriction enzyme, the enzyme could cleave the DNA. if this occurs near a disease-causing gene, the variation (polymorphism = RFLP) can serve as a marker for inheritance of the gene |
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Term
| how are RFLPs still useful? |
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Definition
| clinically in diagnosing duchenne muscular dystrophy |
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Term
| STR / most useful marker for _ because _ |
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Definition
| short tandem repeat = microsatellite repeat DNA marker. found regularly throughout the genome. amplification allows rapid and easy assay - if they're near a disease locus or within it, can be used to track inheritance of the chromosome that harbors the disease gene. / genetic linkage analysis, because the number of repeats is highly polymorphic |
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Term
| what kinds of repeats are in STR |
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Definition
| di tri tetra. CA is most common |
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Term
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Definition
| single base pair differences; like STRs and RFLPs |
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Term
| if DNA marker is 5CM (centimorgan) away from the disease locus, then |
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Definition
| 5% chance for recombination between 2 loci and 95% accuracy of test |
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Term
| first step before linkage analysis |
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Definition
| identify pedigrees where the disease is segregating |
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Term
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Definition
| = linkage of a DNA marker |
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Term
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Definition
| identify the genes we're testing for (corresponding genomic DNA clones) |
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Term
| chromosome level mutations can be detected by (4) |
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Definition
| karyotype, metaphase and prophase analysis, FISH, SKY |
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Term
| tissue types used for chromosome analysis |
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Definition
| any tissues capable of in vitro growth: blood, bone marrow, skin, amniotic fluid, chorionic villi, products of conception, tumor tissue |
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Term
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Definition
| used when you're concerned about a large chromosomal defect - trisomy, monosomy |
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Term
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Definition
| "high resolution" - used when you're concerned about a small deletion or duplication |
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Term
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Definition
| fluorescent in situ hybridization: used when you're concerned about a specific disorder known to be associated with deletion/duplication. velocardiofacial syndrome! 22q11 = lyndsey. used for rapid results for aneuploidy detection, followed by karyotype analysis to rule out other abnormalities |
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Term
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Definition
| Spectral KarYotyping - painting allthe chromosomes with different FISH probes, used to detect rearrangements involving MULTIPLE CHROMOSOMES. simultaneously visualize all the pairs of chromosomes in different colors - fluorescently labeled probes. used for cancer sometimes |
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Term
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Definition
| a relatively small piece of DNA that is used to find another (the target) |
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Term
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Definition
| genes, sections of chromosomes (interphase or metaphase), whole chromosomes |
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Term
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Definition
| denature probe and specimen DNA; hybridize probe to specimen DNA; wash slide in reagent to remove unbound DNA; apply counter-stain and view |
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Term
| indications for prenatal diagnosis (6) |
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Definition
| advanced maternal age, family history of child with developmental delay, ethnic background associated w/ high risk, family history of a genetic condition (AD = 50% risk, AR = 25% risk to each fetus), abnormal ultrasound findings, abnormal serum screening |
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Term
| source of fetal cells (for FISH, karyotype, whatever) / when are these procedures done |
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Definition
| most commonly amniotic fluid. some CVS (chorionic villi samples). / a - 15-16, cvs - 10 weeks |
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Term
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Definition
| ultrasound, nuchal translucency (thickness at back of neck, usually means turner's), serum screening - measures proteins from fetus secreted into mother's circulation |
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Term
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Definition
| CVS, amniocentesis, periumbilical blood sampling, preimplantation genetic diagnosis |
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Term
| advanced maternal age what/why |
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Definition
| more chromosomal abnormalities because the egg remains arrested in prophase I until puberty |
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Term
| advanced paternal age what/why |
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Definition
| more single gene abnormalities = a lot of division = a lot of room for error. |
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Term
| t/f microdeletion syndromes can't routinely be detected |
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Definition
| t. routine amniocentesis only gets major chromosome stuff. if there is a known family mutation, specific testing can happen (achondroplasia, NF-1, tay sachs etc.) |
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Term
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Definition
| preimplantation genetic diagnosis |
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Term
| common prenatal features of chromosomal abnormalities (4) |
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Definition
| spontaneous abortion, positive serum screen, intrauterine growth retardation, multiple congenital abnormalities on ultrasound |
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Term
| common postnatal features of chromosomal abnormalities (4) |
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Definition
| small for gestational age, poor postnatal growth, multiple congenital anomalies (more than three), developmental delay |
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Term
| indications for postnatal chromosome analysis |
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Definition
| dysmorphic features, failure to thrive, developmental delay, short stature, delayed puberty, infertility/miscarriages, family history of chromosome abnormality, mental retardation, advanced maternal age |
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