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26 Cards in this Set
- Front
- Back
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Describe the general structure and organization of the human genome.
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Genome: entire complement of DNA in haploid set of chromosomes of an organism; genomes of some viruses are RNA
---22 autosomes + X/Y sex chromosomes: contain 45-279 million bp (Mb) ---Haploid genome=3.3 billion base pairs DNA/RNA: polymers of deoxyribonuceotides/oxyribonucleotides ---base pairing by hydrogen bonding and base stacking* stabilizes structures and provides specificity of replication, DNA repair, transcription, RNA processing and translation |
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Describe the first generation of DNA sequence analysis.
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Performed by polyacrylamide gel electrophoresis with radioactively labeled NTPs (dideoxyribonucleosides)
---prevents strand extension at 3' end (lack of OH); generates DNA fragments of different lengths Fragments separated by gel electrophoresis: each base has unique identifer ---DNA sequence read directly from bottom of gel upward Had little clinical application |
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Describe the second generation of human genome sequencing.
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Automated sequencing using fluorescent NTPs and various electrophoretic separation methods
---sequence entire genome: took over 13 years and 3 billions dollars |
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Describe the next generation genome sequencing.
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High Throughput Sequencing (THS):
---methods permits sequencing of genome in less than two days for less than $5000; more sequences known, easier it will be to determine other sequences by "referencing" ---direct DNA sequencing will become standard tool in molecular diagnostics |
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Compare and contrast the human nuclear genome with the mitochondrial genome.
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Mitochondrial: circular/duplex
---not all genes necessary for mitochondrial function are in mt genome; most proteins are encoded in nuclear genome |
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Define "gene" and explain the genome complexity conundrum.
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Gene: segment of DNA required to make a function RNA transcript
---coding and non-coding RNA Genome complexity: ---only 1-2% of genome consists of protein coding exons ---exons comprise ~5% of each gene; so protein coding exons + introns only comprise 25% of genome --- ~5000 pseudogenes (non-functional) ---more than 70% of genome is transcribed into RNA ****where is rest of genome?**** ---ENCODE project: goal to find all functional elements of genome: 20% of ncDNA is functional (gene regulation) while additional 60% is transcribed with no known function |
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What are the four classes of noncoding RNAs?
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Translation:
---tRNAs ---rRNAs Processing: ---snRNAs ---snoRNAs Specialized: ---7SL RNA (ER protein export) ---Telomerase RNA (modification) ---Vault RNA (storage) Regulatory: ---small ncRNAs ---siRNAs and miRNAs ---piRNAs ---large ncRNAs ---Xist |
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Describe the microRNAs and their function.
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19-25 nt ncRNAs that bind specific mRNAs through complementary sequences and inhibit translation or cause cleavage (regulate growth and development)
---first discovered in 1993 in C. elegans (worm) ---more than 500 miRNAs have been validated in human genome and total of 800-1000+ are predicted to exist |
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Describe the structure of a metaphase chromosome.
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Chromosomes condensed during metaphase are identified by size, relative position of centromere and G-banding patterns (Giemsa staining)
---most chromosomes contain easily identifiable short (p) and long (q) arms |
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Describe the structure of a metaphase chromatin.
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Chromatin: structural unit of chromosome and is comprised of nucleosomes
DNA is compacted by 10,000 fold in nucleus ---nuclear genome is highly condensed into chromatin around nucleosomes (histone proteins) |
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Define epigenetics and describe the modifications in histones and DNA that lead to epigenetic effects in gene expression.
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Epigenetics:
---heritable changes in gene expression caused by modifications to genome other than changes in underlying DNA sequence ---ex: DNA methylation and histone modification ---can be inherited and are involved in human disease: Prader-Willi syndrome; Angelman syndrome; Beckwith-Wiedemann syndrome; multiple cancers Histone modification: ---level of condensation of DNA has to do with chemistry of histones: favorable interactions=tighter coiling of DNA around istone; less favorable interactions=looser coiling ---Histone (tails) lysines can be acetylated or methylated; serines can by phosphorylated ---modifications provide code from chromatin remodeling (gene expression/silencing) DNA Methylation: ---of cytosine, marks genome (CG sequences) ---patterns maintained in newly replicated DNA: maintenance DNA methyltransferases methylate hemimethylated C residues during replication ---methylation turns off genes by attracting silencing complexes to block RNA polymerase Demethylation: 5mC erasure ---can occur via several pathways by going through intermediates (complicated) |
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Describe genomic imprinting.
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Imprinted genes are protected from DNA methylation erasure
---less than 100 imprinted genes have been identified in humans ---located throughout genome including in X chromosome ---often associated with growth and development Can cause selective silencing of one parent's gene if offspring differ in allele of gene to be expressed |
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Describe the human microbiome.
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100 trillion human cells in human body
---1000 trillion microbial (viral, bacterial, protozoan, fungal) cells in human body, incl. over 600 different species of bacteria Optimal balance of microbes is critical in human health ---found in nasal, oral, skin, gastrointestinal and urogenital areas Enteric bacteria: ---over 1000 bacterial species inhabit gut, with anaerobes comprising 99% ---60% of dry mass of feces ---function: digestion of unused energy substrates, fermentation, training of immune system, production of vitamins/hormones ---perturbation of symbiotic relationship with host causes disease of digestive track: diarrhea to inflammatory bowel and cancer; implicated in obesity, diabetes, metabolic syndrome ---overgrowth of E. coli, Salmonella typhimurium and C. diff can cause serious illness |
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Describe the relationship between DNA molecule and chromosomes.
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46 chromosomes: diploid (all except sex chromosomes X/Y)
---normal dosage |
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Describe chromosome disorders.
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Chromosome disorders: < 1%
---abnormalities in number or structure of chromosomes; alterations affect genes on chromosomes ---too many, too few, altered genes ---polyploidy and aneuploidy (change in one) ---insertions, deletions, and translocations |
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Describe single gene disorders.
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Single gene disorders: 2%
---exact number of genes in human genome approx. 20,000-25,000; typically code for proteins, but some code for RNA ---alterations in coding sequence produce effects on function of protein - loss (typical), gain, alteration of function ---single genes are basis for Mendelian inheritance: autosomal disorders and sex-linked disorders |
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Describe mitochondrial disorders.
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Mitochondrial disorders: ~1:5000
---mitochondria are organelles responsible for most importantly production of ATP; have own genome ---is disrupted by same mutational mechanisms as nuclear DNA ---non-Mendenlian inheritance: maternal inheritance for mutations in mtDNA ---Mendenlian inhertiance for mt genes on nuclear DNA |
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Describe multifactorial disorders.
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Multifactorial disorders: common
---simple congenital malformations: 1% ---common disorders of adults: 60% Combination of genes and environment ---polygenic: many genes acting together ---teratogenic: primarily environmental based ---multifactorial: combination |
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Describe the different inheritance patterns.
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Identified through 3-generation pedigrees
---for single gene disorders - autosomal dominant or recessive, X-linked recessive or dominant (uncommon) |
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Identify chromosome analysis through karyotypes.
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Karyotypes:
---detect alterations in number, large duplications/delections, translocations Ex: Trisomy 21 - aneuploidy: abnormal number of chromosomes (dosage problem); leads to dysmorphism |
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Describe chromosome analysis using FISH and CGH.
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Fluorescent in-situ hybridization and comparative genomic hybridization
---detect small deletions and duplications (CGH) FISH: uses control probe and probe for area of interest ---if probe hybridizes to sample, DNA sequence is present CGH: stick DNA to probe; looks at whole genome, don't need to be as specific ---too much/duplication-green dots (patient DNA) ---not enough/deletion-red dots (control DNA) ---normal-yellow dots |
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Describe chromosome analysis using genome sequencing.
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Exome sequencing:
---determination of sequence of coding portion of genome; mutations that cause disease will likely alter this coding sequence Whole genome sequencing: ---sequenc everything; will pick up mutations in promoter regions, introns, 3' untranslated regions Problems: --all carriers for many diseases ---detect diseases with future consequences ---sequence variation of unknown clinical significance |
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Describe biochemical tests for genetic diagnosis.
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Use of tools of chemistry to look for abnormal products of metabolism or abnormal proteins
---uses techniques such as gas/liquid chromatography ---enzyme assays ---newborn screening (looks for things like phenylketonuria - leads to accumulation of phenylalanine). |
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Describe DNA diagnostics for genetic diagnosis.
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Direct mutation analysis:
---DNA sequencing ---small DNA probes that hybridize to mutated DNA Linkage analysis: ---utilization of trackable gene markers next to gene of interest DNA fingerprinting: ---analysis of variation of sequence of genome to identify individuals ---CSI, paternity |
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Describe non-Mendelian inhertiance.
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Mosaicism: more than one genotype in person
Mitochondrial inheritance: abnormal mt passes on in cytoplasm of ovum from mother Genetic imprinting: parent-of-origin difference in gene expression Uniparental disomy: both chromosomes of pair inherited from one parent Unstable triplet repeat mutations: develop by expansion of normally present trinucleotide repeats |
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Describe the ethical issues surrounding genetic testing.
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1. Insurance operates on knowing general risk of developing disease
---can alter insurance risk if do DNA test to tell is develop disease 2. Two categories: ---Presymptomatic: testing person for genetic mutation that will certainly cause disease later in life ---fully penetrant genes ---offered to children only when medical benefit; to adults after informed consent ---Predisposition: testing person for genetic mutation which will possibly cause disease later in life ---causative genes are incompletely penetrant 3. Unique privacy issues: other family may be at risk 4. May stigmatize/cause discrimination 5. Access to counseling not widely available: for detection of false-paternity, stigmatization (survivor guilt), loss of employment/insurance; psychological harm |
