Molecular Genetics Lab Quiz 3

Alcohol Dehydrogenase (ADH)

Enzyme involved in ethanol metabolism

ADH Expression

More produced in ethanol, males head

ADH Lesson

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

ADH Organism

Drosophila melanogaster

ADH Methods

Cellulose acetate zymogram

ADH Male vs. Female

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

RAPD PCR Abbreviation

Random Amplified Polymorphic DNA; Polymerase Chain Reaction

RAPD PCR Use

PCR technique used to amplify random unknown sequences

RAPD PCR Primer

One nonspecific primer

RAPD PCR Results

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

RAPD PCR Methods

PCR, gel electrophoresis

RAPD PCR Results Interpretation

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

Traditional PCR

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

Central Dogma

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

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

Transcriptome

all RNAs in a cell (noncoding & coding)

RNA-Seq Analysis

1. Extracted mRNA is reverse transcribed into cDNA

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

RNA-Seq Reason

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

Histograms

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

Histogram Introns

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

Histogram Exons

Higher number of reads

Isoforms

Difference in peak height can indicate alternative splicing effects

Male vs. Female Tracks Identification

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

Viewing exon-intron boundaries

1. Zoom in

2. Set Screen to 15-20 nts

3. View nts & positions shown in window

Making a picture

1. Draw exons and introns

2. Label boundaries with the nucleotide positions from genome browser

3. Label splice donor/ acceptor sequences

Boxes

Exons; sequenced mRNA

Line

Introns

Exon junction

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

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)

Module 5: Translation

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

Reading Frame: Gene Transcription direction (- or +)

6 possibilities:

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

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

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

Coding DNA Sequence

Coding portion of exons

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

Module 6: Alternative Splicing

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

Alternative Splicing Reason

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

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

RNAi Organism

Caenorhabditis elegans- roundworm or nematode

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