Lecture 6

Question 1

The main difference between DNA analysis and gene expression analysis

Answer 1

Introns.

Question 2

cDNA library

Answer 2

Collection of cDNA molecules formed by reverse transcription of post-splicing mRNA

Question 3

How and why is plasmid used in formation of cDNA library?

Answer 3

How: Plasmid contains origin of replication, ampicillin resistance gene, target gene, and multiple restriction enzyme sites. Heat shock and CaCl2 induce bacteria to take up plasmid. Ampicillin ensures that only bacteria that were transformed (took up plasmid) survive. These bacteria and their plasmids replicate.

Why: Bacteria are really good at making more of themselves and their plasmids.

Question 4

Reverse transcription for creation of cDNA library (5 steps)

Answer 4

mRNA with oligo-dT primer on poly-A tail

Reverse transcriptase, then remove RNA with alkali and add poly-dG tail

Add oligo-dC primer on poly-dG tail, then DNA polymerase

Protect cDNA by methylation at internal EcoR1 sites

Add EcoR1 sites to ends of cDNA, then ligate to plasmid

Question 5

To detect full-length gene in plasmid (3 steps):

Answer 5

Transfer colonies to nitrocellulose filter

Denature plasmids

Hybridize with probe

Question 6

Gene expression analysis tool (DNA level)

Answer 6

Southern blot

Question 7

Use of Southern blot in diagnosis of fragile X syndrome (5 facts)

Answer 7

Gene has (CGG) x n between 2 restriction (cutting) sites

Normal gene has n=30 CGG repeats

Methylation induced by high n (n>250 in mutated gene)

Methylation also covers EagI restriction site ==> gene not cut here

Mutated gene shows long segment on blot; normal gene was cut at EagI site

Question 8

Gene expression analysis tool (mRNA level)

Answer 8

Northern blot

Question 9

Advantage of Northern blot over Southern blot

Answer 9

Quantitative analysis (higher gene expression = more mRNA = stronger signal on Northern blot)

Question 10

Microarray (3 facts)

Answer 10

Tool used to analyze expression of >8,000 genes

Two labels (red and green) enable measurement of gene expression in normal and disease states (heatmap)

Useful in diagnosing diffuse large B-cell lymphoma (DLBCL) and non-Hodgkin’s lymphoma, and in estimating disease prognosis

Question 11

First step in protein analysis

Answer 11

Use SDS (a detergent) to disrupt hydrogen bonds and hydrophobic interactions, and use a reducing agent (e.g. 2-mercaptoethanol) to break disulfide bonds

Question 12

Gene expression analysis tool (protein level)

Answer 12

Western blot

Question 13

Two methods of protein separation

Answer 13

2D gel (one dimension is charge, the other is size)

Column chromatography

Question 14

Three types of column chromatography

Answer 14

Gel filtration- proteins filter around polymer gel beads that have divots; large proteins filter out faster because they can’t get caught in the small divots

Affinity- proteins filter around polymer gel beads that have antibodies; proteins that bind to antibodies are eluted later with pH 3 fluid

Ion exchange- proteins filter around polymer gel beads that have + charge; negatively-charged proteins eluted later with salt solution

Question 15

Proteomics

Answer 15

Large-scale analysis of proteins

Question 16

Mass spectrometry

Answer 16

Tool used to determine identity and sequence of proteins

Question 17

Technique of Western blot (4 steps)

Answer 17

Transfer polyacrylamide gel electrophoresis results to different membrane

Incubate with first antibody (monoclonal); wash excess

Incubate with enzyme-linked second antibody (polyclonal); wash excess

React with substrate for second antibody for chromogenic detection