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12 Cards in this Set
- Front
- Back
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mRNA makes up only ____% of the genome. The rest are tRNAs and rRNAs.
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5
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3' UTRs are
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Variable in length, sometimes contain regulatory elements, influences life of transcript and protein levels
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How does one make ESTs/cDNA?
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extract RNA, purify mRNA, reverse transcription into DNA, clone into a vector, sequence from one end to get either 5' or 3' cDNAs
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What are 5' ESTs useful for?
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Gene identification - 5' either begin with the coding sequence of the mRNA, or start at the true 5' end of the mRNA, crossing the short 5' UTR
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3' ESTs are useful for
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Gene enumeration. There is usually a bias towards the 3' end in DNA synthesis.
-Start at the polyA tail. |
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Normalizing a cDNA library is based on
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hybridization kinetics: common sequences rehybridize more quickly and are removed.
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What are some advantages to microarrays?
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Parallelism, miniaturization, automation (manufacture and processing), multiplexing
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Uses of microarrays?
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-analyze gene expression (probes complimentary to cDNA
-genomic hybridization (detect features of genome) -SNP detection -protein microarrays |
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What investigations may be done using microarrays?
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-investigate cellular states and processes (eg over time)
-Compare related species or strains -healthy vs diseased individuals or tissue comparisons -determine gene function |
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WHAT ARE THE ADVANTAGES OF THE DIFFERENT MICROARRAY PROBES?
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short oligonts (25nt):
-high specificity -small spot size -disadvantages: low sensitivity, expensive long oligonts (50-75nt): -balance specificity and sensitivity -disadvantages: larger spot size, expensive large probes (PCR products or cDNAs) -higher sensitivity, cheaper to produce -disadvantages: lower specificity, allows cross-hybridization |
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Genes that cluster together based on their expression across samples/conditions may be
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1) regulated by similar genes
2) involved in similar processes |
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Ways to quantify gene expression
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Hybridization-based approach
-microarrays Sequence based approach -counts from cDNA/EST libraries, Sanger sequencing -RNA-seq next generation sequencing |
