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158 Cards in this Set
- Front
- Back
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What would you expect a mitochondrial inherited disease to look like?
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Passed from mother to son or daughter… but not inherited from the father
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What is the DNA capacity of BAC and where was it used?
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150-200 kb… useed to sequence to the human genome.
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what is a proteome and how many do humans have?
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protein coding genes: 3-40 thousand
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Are polymorphic genetic markers evenly spaced? How were the markers make?
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yes, they were made from microsatellite simple tandem repeat sequence such as (CA)n…
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What is the function of the microsatellite simple tandem repeat sequences?
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none, but they are helpful in mapping
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Where are PCR primers generated from?
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PCR primers can be generated from unique sequences flanking the ese sequences.
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What do you find at CpG islands?
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transcription initiaton site of a genes (CpG often the site of methylation)
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Where are most rRNA genes?
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on the p arm (acrocentric)
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What do small nuclear RNAs do?
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they form ribonucleoprotein particles and are important for splicing
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What percentage of the genome is exons?
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1%
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what is the typical length of a gene?
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10-15 kb including exons and introns
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what is the average length of an mRNA?
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2.5 kb
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How were mRNA from tissue isolated in the genome?
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They were reversed transcribed to cDNA and then shotgub subcloned into bacterial vecdtors
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What do you call a copy of a gene containg its normal intron/exon structure, but harboring mutations, due to time and lack of selection?
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unprocessed or pseudogene
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Can pseudogenes be expressed as mRNA?
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yes, but because they don't produce a functional gene they are not translated.
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Where do you find pseudogenes? What purpose do they serve?
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they cluster in the pericentromeric or subtelomeric regions… where they serve as a sink or donor of new genes or dying genes.
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Where do psuedogenes come from?
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they are defective (incomlete) gene copies that result from the reverse trancription of mRNA to a cDNA in a cell followed by its reinsertion randomly into the genome
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What percentage of the human genome is made up of transposable elements contain?
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50%
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What do transposable elements comprise?
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high copy reptetive DNA elements, ie., LINEs and SINEs (long and short interspersed nuclear elements)
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Do LINEs function? If so, what is required?
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a small number are functional and can transpose… b) 2 ORFs one encodes for a reverse transriptase and endonuclease wich make it possible for the element to jump around in the genome
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What is an HERV?
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it's an endogenous human retrovirus… most copies are non-functional
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What is the most abundant SINE? Where are they found?
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Alu (millions of copies)… found in gene rich areas, which suggests a functional role
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What are the hazzards associated with LINEs and SINEs?
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they can interrupt a gene, or unequal crossover between similar SINEs and LINEs can cause chromosome rearrangements like delitions or translocation that result in disease
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Why should we care about noncoding junk DNA?
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they can be used as markers, for example microsatellites which can help identify candidate genes.
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What is the advantage of possessing the sequence from other species?
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can be used as models, identification of similar (conserved genes) and used for tests
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What makes us human vs. non-human?
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?
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Are reuglatory regions usually well conserved?
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Yes
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Of the non-coding genes what are the two catagories of genes?
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pseudogenes and regualtory regions
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besides genes, what is in the geneome?
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junk DNA
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Is there is a positive relationship between the number of exons and importance of the gene?
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no
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What is the RB1 gene and what can happen if it's mutated?
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it's a tumor repressor gene… if mutated it can lead to retinal tumors
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What direction is the U16 gene on the retinobastoma gene transcribed?
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its 5' to 3', but it's coded for on the opposite strand… thus genes can be oriented in forward or reverse direction but they are always transcribed 5' to 3'
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Are genes that are related always together? Give an example of close and distal
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no, some families of are distal… e.g., close: ß-globin… distal: histones
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What are referred to as non-coding coserved regions?
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transcriptional regulatory regions… since these genes are well conserved
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What can we use to find whole genes?
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comparative genomics (genes from different species)
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What can be done to determine the function of gene?
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produce KO mice to see if it's essential to life
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review page 27
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What can a centromere be used for in working with DNA?
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Centromeres are characterized by repeats and can help orient 10 Mb region
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What does UTR stand for?
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untranscribed region at the 5' and 3' regions of a gene
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What are unique variations in DNA?
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polymorphisms… satellites and SNPs, which are not in the coding region and thus harmless, but can effect disease.
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How many microsatellites do you find per 50,000 bp?
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one
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Are microsatellite similar in related individuals?
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no, the are highly variable if you are not related
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Are microsatellite evenly spaced?
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no… they are used as markers in linkage studies
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Can SNPs be important to function?
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yes… they are single base pair variations that differe from one person to another, within a family… they can be in the gene or in regulatory regions… Anywhere.
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What are SNPs used for?
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positional cloning
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At what frequency do you find SNPs per 1000 bp?
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one… less than 1% of genome… most are found in the coding regions
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What's a non-synonymous SNP?
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codes for a change in codon
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Are most SNPs in coding or non coding genes?
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non coding, but they can still effect disease by effecting regulatory regions
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Where are genes located, light or dark regions?
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light
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Where are LINEs located, light or dark regions?
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dark
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Lecture 2
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When do Chromosomal Disorders have a great impact?
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before birth
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When do single gene disorders have a great impact?
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early childhood
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When do multifactorial disorders have a great impact?
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later in life in adulthood
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What is cytogenics?
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A method by which chromosomes can be analysed under the microscope… for example to find trisomy
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What is a bipartite?
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its when DNA consists of 2-sister chromatids with exact DNA
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When is the reduction stage of meiosis? Why?
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MI, because the 2-sister chromatids stay together (it's like 2 of the same DNA, so it reduced from a diploid to a haploid)
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When does homologous recombination occur?
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MI: meiotic cross-over pachytene stage) before the sister chromatids separate
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In what stage do oocytes become arrested?
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prophase I, until ovulation
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What test is used to check for chromosomal abnormalites?
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G banding of lymphocytes of blood
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What 2 tests are used to detect SUBTLE rearrangements or terminal deletions?
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Fluorescence in situ hybridzation (FISH)
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What information is needed for FISH?
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you have to have an idea of what abnormality you're looking for, with respect to disease (and or a particular deletion or duplication)
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What is the kb size limitation?
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10-20 kb
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Describe FISH protocol
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a single stranded labeled DNA probe is hybridized to the region of interest
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What test can help determine whether or not a trisomy event is due to mother or father contribution?
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polymorphic microsatellite markers by P"CR approaches, where gel electrophoresis of microsatellite markers on chromosome 21 (3 each) the DS child is run and compared with the mother and father's microsatellite… note that the one that contributed 2 is the contributor of the extra chromosome
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What is the frequency for aneuploidies?
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1/154 live births
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Which 3 chromosomes are implicated in trisomy events most common trisomy
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21 (DS), 18 and 13
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What is Turner's?
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45 X females
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What is the cause of an unbalanced translocation?
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these children are the offspring of partial aneuploidy events of a normal carrier of a BALANCED TRANSLOCATION
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What congenital effect does an altered gene dose usually result in?
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mental retardation
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What is a contiguous gene disorder?
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it is multiple haploinsufficiency caused by the small hemizygous losses of DNA (on the order of 15-30 genes)
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What is the usual cause of unbalanced translocation?
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crossover events between region specific LCR (low copy repeat) on chromosomes.
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What is used to determine an genomic disorders caused unbalanced translocation?
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FISH
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When is interphase FISH mapping used ?
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to detect multiple duplication
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What type of deletions are associated with mental retardation?
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terminal deletions, as they are near the telemers.
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When do male gametes undergo prophase I?
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in adulthood
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What is occuring during prophase I?
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segregation… haploids are created
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When does recombination occur?
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MI
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What is the difference between metaphase I and metaphase of meiosis?
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MI produces haploids that may be different due to cross over… mitosis metaphase produces daughter cells (diploid)
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What do defects in meiosis lead to ?
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aneuploidy
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What is the most common cause of mental retardation?
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aneuploidy
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What is the frequency of Down Syndrome?
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1/800 live births
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How many different (not alike) alleles for a given chromosome do you find in a Down Syndrome individual, where non-disjunction occurred in MI?
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M1 = 3 Types
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How many different (not alike) alleles for a given chromosome do you find in a Down Syndrome individual, where non-disjunction occurred in MII?
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MII = 2 different types
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What is a haplotype?
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the unique order of markers along a chromosome
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A child is born with major birth defects (craniofacial, heart, brain), severe developmental delay. What diagnostic tests would you do?
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1. Do a kayotype oto see if chromosomes are abnormal in sturcture or copy number… 2) If you suspect a specific genetic syndrome, use a molecular cytogenetic probe and perfomr FISH mapping to see the deleted or duplicated region
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At what phase in mitosis would you do a FISH test?
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Metaphase, where the chromosomes are condensed.
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What test would you use to detect multiple rearrangements, such as seen in cancer?
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SKY (spectral karyotyping)
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What are genomic disorders caused by?
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deletions or duplications
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What causes VCFS (di George Syndrome)?
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deletion of 22q11.2
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What causes William's syndrome?
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deletion of part of 7q11.2
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Besides learning disabilities and characteristic faces, what do di George people present with?
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heart defects and immune disorders
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What test do you use to diagnose di George or William's syndromes?
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FISH
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What is the mechanism responsible for getting deletions or duplications in the genome?
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Non-allelic homologous recombination events between chromosomes cause deletions or duplications
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Is it just as possible for a deletion or duplication to occure between two different alleles (inter-chromosomal as it is for this to occur on the same chromosome?
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yes, Intra-chromosomal
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Are most parents normal in genomic disorders?
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yes
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Lectures 3 and 4
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In what order does the protein made (Hint: direction 3-5, C-terminus... or 5-3, N-terminus)
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5' -> 3' N-terminus
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What is the % of introns and exons/
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exons = 5%… introns = 20%
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What percentage of the human genes are alternatively spliced?
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40%
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Which restriction enzyme is blunt?
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Hpal
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What is the average size of a 4 bp long restriction enzyme?
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256 = 4*4*4*4
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Does the restriction enzyme cut uniformaly?
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no, it's an average
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What does a DNA ligase do?
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it covalently links two pieces of DNA
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What happens when you place a restriction enzyme in with DNA and a plasmid and then ligase it?
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recombinant DNA, where the DNA of interest is inserted into the plasmid
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What are preferred properties of cloning vectors?
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minimal size, origin of replication, high yield of DNA, dominant selectable markers (like AMP or tricylin), lots of restriction sites, selection screen for inserts, inducible or high level of expression)
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What is the protocol for Cloning a gene of interest?
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place DNA in container with the vector and Restriction enzyme… 2) add ligase… 3) insert into bacterial cells… 4) grow on selective plate with antibiotic… 5) bacterial replication will reproduce your sequence
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What does forced dimerization of Caspase-8 cause in adiocytes?
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apoptosis
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What is the result transfection with the FAT-ATTAC gene in mice?
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decreased obessity
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How do you make a cDNA library? (5 steps)
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1) mRNA is prepared from the tissue of interest… 2) a complementary copy of DNA (cDNA) is made using oligo-dT as a primer and reverse transcriptase as a primer… 3) The RNA is removed using RNAseH, which digests the RNA in RNA-DNA hybrids… 4) a second strand of DNA is made using DNA polymerase... 5) The ds DNA can now be inserted into a vector
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What is cDNA?
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it's a sequence of DNA made from mRNA
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Why would you make cDNA?
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Since it's made from mRNA you know that it's the gene that was being translated… " cDNA: gene representation is relative to expression level"
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How is a Genomic Library made?
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by purifying DNA from a human tissue or cell line… cutting it with a restriction enzyme… then ligating the resulting fragments into a vector
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What does the relative abundance of cDNA represent?
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the actual presence in the cell
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What is nucleic acid hybridization?
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anealing of two strands of DNA-DNA or DNA-RNA
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How do you screen a DNA fragment by hybridization?
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Lyse phases (vector), and denature DNA --> hybridize to labeled probe
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What does Gel electorphoresis do?
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separates DNA and RNA by size
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What drives the molecules throught an electrophoresis gel?
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current
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Which flow through an electrophoresis gel, large or small particles?
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small
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What is Southern blot used to measure?
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DNA
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What are the 4 steps in Southern blotting?
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1) restriction digestion of DNA… 2) Gel electrophoresis of DNA by size… 3) Transfer to membrane… 4) use a labeled DNA probe for detection
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What is the DNA tranferred to from the agarose gel?
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Ntrocellulose paper
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What is RFLP used for?
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genetic finger printing and paternity cases
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What is a RFLP?
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restriction fragment length polymorphism
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what is a missense mutation? What causes it?
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codes for a different amino acid… substitution
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what is a nonsense mutation? What causes it?
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codes for a stop codon (UAG, UAA, UGA)… substitution
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what is a frameshift mutation? What causes it?
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when all subsequent amino acids are wrong… deletion or insertion… usually fatal
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what is a loss of codon mutation? What causes it?
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an entire amino acid is lost… deletion
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what is a duplication mutation? What causes it?
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when an entire sequence is duplicated… (transposable elements)
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What is a trunctation usually caused by?
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nonsense mutation or frameshift
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Is a truncation usually a dominant disorder or a recessive disorder?
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recessive
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Is a truncation mutation loss or gain of function?
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loss
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What is a dominant negative mutation?
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when an abnormal protein phenotype poisons the other functional protein
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What is haploinsufficiency?
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When 50% of the gene product is insufficient to support normal function
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Is a haploinsuffiency usually recessive or dominant
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dominant
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What is a gain of function mutation?
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where an altered protein has a new property (can be an increase in normal or different)
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LOOK UP GAIN OF FUNCTION
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What does a Northern blot measure?
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RNA expression
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What is the Northern blot protocol?
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Electrophoresis of RNA… 2) transfer to membrane… 3) use a labeled probe for detection
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When is a Northern blot used?
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to check expression of a gene given a stimulous over time… mRNA
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What is the expression of mRNA Acrp30 associated with?
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fat cell protein
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What does the overexpression of adiponectin lead to ?
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increased fat mass
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What would you use to measure expression of a mRNA (or DNA) in a tissue?
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in situ hybridization
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What is the protocol for PCR?
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add DNA of interes… 2) add primers… 3) heat to 90°… 4) cool to aneal… repeat…
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The next question related to the use and protocol of microarrays
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What do each spot of a microarray have on it?
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ssDNA probes for thousands to milions of genes, each spot with a location specific to a particular gene
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After you obtain mRNA from the tissue sources being compared (e.g., treated and untreated with drug… diseased vs. normal), WHAT DO YOU DO WITH THE mRNA NEXT?
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Convert to cDNA and add a fluorescent label for each one (red and green)
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What do you do with the cDNA?
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apply to the chip… the cDNA and the probe will hybridize
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What does it mean when the cDNA binds to the chip?
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The binding corresponds to the expression of that gene being expresson in the tissues (differentially red or green)… a computer will analyize these expressions
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What is the benefit of using microarrays?
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many fragments can be visualized at one time
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What is laser capture microdissection (LCMD)?
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laser cuts and collects cells in eppendorf
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What is a Western blot used for?
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protein analysis
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What is the protocol for SDS-page Western blots?
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1) SDS-page & heat: denatures and normalizes the charge on proteins to NEGATIVE CHARGE (because not all the charges are the same on the same)… 2) place in polyacrylaamide gel-electrophoresis… small protein will travel faster…
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How can you use isoelecdtric focusing to higher resolution of SDS-PAGE?
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it will move along the gel to its isoelectric point… THUS a protein can be analyzed by size and isoelectric focusing… 2-D SDS-PAGE… this is a better purification (resolution)
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How do you vizualize the the proteins in an SDS-PAGE Western blot?
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apply a porous sheet (nitrocellulose) to the gel… apply an antibody for the protein… was away excess proteins (which have not bound to the probe)… incubate with an enzyme linked antibody that binds to the other end of the probe.
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Adiponectin is an example of a use for SDS-PAGE Western blotting. What does lowered high molecular weight of Adiponectin in individuals predict?
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insulin resistance and metabolic syndrome
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What can you use to visualize where a protein is being expressed within a cell?
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Immunofluorescence
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What can you use to sequence protein amino acid sequences?
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mass spectrometry
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