Lecture 21 Class: Biotechnology
Starter:
Fragmenting DNA or RNA for sequencing requires which reaction?
A. Dehydration
B. Hydrolysis
Comparison of biotechnologies:
Technique
Restriction enzymes
Sanger sequencing
Gel electrophoresis
Probe for TTTT sequence
Mass spectroscopy
Next-Gen sequencing
Match
Purpose
Sequences full genomes
Binds to mRNA
Measures mass of peptide fragments
Cleaves DNA at a particular sequence
Sequences DNA by halting replication with a
fluorescent nucleotide
Separates DNA or protein fragments by size
Reading a Gel:
All three wells were filled with the same DNA and different restriction
enzymes.
1. Which fragment is smaller, 2 or 3?
A: 2
B: 3
2. Fragment 1 is 500 base pairs long. Which is 450 base pairs?
A: 2
B: 3
3. How many places did a restriction enzyme bind in Sample B?
A: 1
B: 2 C: 3
Which of the following would move further down an electrophoresis gel?
A. A small cytoplasmic protein
B. A small integral membrane protein
Lecture 20 Page 1
Current research using restriction enzymes:
Kitano, K., et al. (2011), CpG island methylation of microRNAs is associated with tumor size and
recurrence of non-small-cell lung cancer. Cancer Science, 102: 2126–2131.
Background:
Methods:
Amount of miRNA methylation was measured in patients with different cancer severities
Restriction enzymes chop up chromosomes, kept DNA bands associated with miRNA that breaks
down mitosis “go” mRNA. Measured methylation quantity with electrophoresis.
Lecture 20 Page 2
Results:
Representative combined bisulfite restriction analysis results of non-small-cell lung carcinoma tissue
samples. Arrows, unmethylated alleles; arrowheads, methylated alleles; L, DNA size markers;
numbers on top, tissue samples; stars, samples with significant restricted fragments from methylated
alleles.
Current research using DNA sequencing:
Ramagopalan, SR et al. 2010. A ChIP-seq defined genome-wide map of vitamin D receptor binding:
Associations with disease and evolution Genome Res. 20: 1352:1360.
Background:
Vitamin D is able to cross the membrane and binds to a complex that activates
transcription.
Lecture 20 Page 3
Methods:
Findings:
• __________ binding sites for Vitamin D
•
•
Over ___________ genes change expression
Affected genes include those associated with multiple sclerosis and Crohn’s disease
Sample Problems:
When might a transcriptome indicate that a mRNA is present, but a proteome not find any evidence
of the protein?
A. RNA was alternatively spliced
B. Protein was immediately ubiquitinated
C. Transcription was not regulated by a transcription factor
You are interested in whether a family of three who all have irritable bowel syndrome have the same
mutation in a regulatory gene. Which is necessary for restriction enzymes to be a useful tool?
A. The mutation must produce a changed protein
B. The mutation must be within a restriction site
C. The mutation must produce a different mRNA molecule
Formaldehyde is a chemical that binds protein to DNA. A good use of this chemical would be to:
A. Compare restriction fragments of hemoglobin alleles
B. Look for genes that are associated with a transcription factor
C. Compare proteins that are expressed in patients who are being treated for cystic fibrosis
Lecture 20 Page 4