Molecular Genetics Lab Quiz 3

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39 Terms

1

Alcohol Dehydrogenase (ADH)

Enzyme involved in ethanol metabolism

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2

ADH Expression

More produced in ethanol, males head

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3

ADH Lesson

Gene expression can be influenced by factors in the environment, gender, and body part

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4

ADH Organism

Drosophila melanogaster

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5

ADH Methods

Cellulose acetate zymogram

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6

ADH Male vs. Female

M: smaller, fuse bands; F: bigger, separate bands

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7

RAPD PCR Abbreviation

Random Amplified Polymorphic DNA; Polymerase Chain Reaction

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8

RAPD PCR Use

PCR technique used to amplify random unknown sequences

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9

RAPD PCR Primer

One nonspecific primer

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10

RAPD PCR Results

multiple bands in gel electrophoresis results because primer binds to multiple sequences

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11

RAPD PCR Methods

PCR, gel electrophoresis

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12

RAPD PCR Results Interpretation

Comparing bands in different lanes can indicate whether they are genetically similar

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13

Traditional PCR

Uses forward and reverse primers to amplify known DNA sequence and results in one band on gel electrophoresis

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14

Central Dogma

DNA transcribed into RNA (transcriptome). Its then translated into a protein.

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15

Module 4: Splicing

Process in which introns are removed from pre-mRNA via the spliceosome to produce mature mRNA molecules; Mature mRNA is the template for protein synthesis

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16

Transcriptome

all RNAs in a cell (noncoding & coding)

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17

RNA-Seq Analysis

  1. Extracted mRNA is reverse transcribed into cDNA

  2. Fragments of cDNA are then sequenced and mapped back to the genome

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18

RNA-Seq Reason

Analyzing mRNA molecules allows us to analyze transcription regulation/gene expression

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19

Histograms

Y-axis: # of reads that map to the position on genome; X-axis: position of genome

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20

Histogram Introns

Gaps of gene expression because they're spliced out from mature mRNA that's sequenced.

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Histogram Exons

Higher number of reads

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22

Isoforms

Difference in peak height can indicate alternative splicing effects

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23

Male vs. Female Tracks Identification

Differential gene expression, sex-specific isoforms, Exon-Intron boundaries counts, Exon usage/Splice site variations

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24

Viewing exon-intron boundaries

  1. Zoom in

  2. Set Screen to 15-20 nts

  3. View nts & positions shown in window

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Making a picture

  1. Draw exons and introns

  2. Label boundaries with the nucleotide positions from genome browser

  3. Label splice donor/ acceptor sequences

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Boxes

Exons; sequenced mRNA

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27

Line

Introns

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28

Exon junction

RNA fragment sequence matches that of DNA downstream. Why? Bases in between must have been removed

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29

Non-randomized Splicing

Machinery recognizes specific sequences at the beginning and end of introns.

Splice donor: 1st two nucleotides of intron (GT/GU)- Splice acceptor: last two nucleotides of intron (AG)

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Module 5: Translation

Process of converting mRNA molecule sequence to an amino acid sequence that encodes for protein via the ribosome

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31

Reading Frame: Gene Transcription direction (- or +)

6 possibilities:

(-), Forward; (-), Reverse; (+), Forward; (+), Reverse; Direction (+); Direction (-)

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Reading Frame: Start codon where?; Stop codons in exons?

Exons can have different reading frames; Stop codons in UTRs are okay

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Coding DNA Sequence

Coding portion of exons

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Phases: codon cut off by splice site?

  1. 1 base long partial codon at the end of the exon- Couldn’t be complete

  2. 2 base long partial codon at the end of the exon- May be compensated for

  3. Complete codon before splice site- Normal translation

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35

Module 6: Alternative Splicing

Exons of the same gene are joined in different combinations to produce different proteins

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36

Alternative Splicing Reason

mRNA allows us to be adaptable to environment and through development. We need diverse proteomes to meet our needs

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RNAi (RNA interference)

Small RNA molecules guide endonuclease activity to degrade mRNA transcripts. Degraded transcripts cannot be translated therefore their proteins are not expressed. This results in observable phenotype

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38

RNAi Organism

Caenorhabditis elegans- roundworm or nematode

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39

RNAi Experiment

C. elegans is fed E. coli with DPY( dumpy determines fat/ short phenotype) gene which results in phenotypic change of shorter C. elegans

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