Molecular Technique

Question 1

Macromolecules in Cells

Answer 1

Nucleic Acids
Proteins
Lipids
Polysaccharides

Question 2

Molecular Techniques Often Used in Developmental Biology

Answer 2

Purpose:
Find your gene of interest or its product
Determine if your gene of interest is transcribed and/or translated
Understand the expression pattern of your gene of interest

Techniques commonly used:
In Situ Hybridization
Reverse Transcription - Polymerase Chain Reaction (RT-PCR)
Expression Profiling: Microarray
Western blot

Question 3

In Situ Hybridization

Answer 3

It can be used to:
Determine where & when specific mRNA is expressed
Identify gene on a chromosome

Signal detection:
 Colormetric probe (Enzymatic, Alkaline Phosphate)
 Radioactive probe (Film)
 Fluorescence probe (FISH)

Usually mRNA.
Where on a chromosome a gene is

Question 4

In Situ HybridizationWhere and When Specific RNA is Expressed

Answer 4

Colormetric

Mouse E10.5

RNA Probes:
Phox2a
En1
Uncx4
Lmx1b

Whole Mount In Situ Hybridization (WISH)

Phox2A – expressed in the dorsal ganglion regions of the nervous system
EN1 – expressed in the vertebrae and the internal gut
Uncx4- expressed in the brain and vertebrate and spinal cord
LMX1b – expressed in the brain, spinal cord and hind limb

(alkaline phosphatase)

Question 5

In Situ HybridizationWhere and When Fig(alpha) is Expressed in an Ovary

Answer 5

Radioactive:
Sections through mouse ovary

Probe = Radioactive Anti-sense RNA to Fig a

Where is Fig (alpha) located?

Visualized on a film
Cluster of the white is the fig alpha and is expressed in the primary oocytes
Bright white is probably the primordial follicle

Knockout does not show any expression

Question 6

In Situ HybridizationHow many copies of a specific chromosome are in a cell?

Answer 6

FISH: fluorescence in situ hybridization

Used here to determine number of copies of chromosome 21 in a cell.
Cell has been treated with a fluorescent probe that binds to a gene on chromosome 21.
How many copies of chromosome 21 are in each cell?

FISH technique stains with DAPI as well

Blue stain on nuclei, different color used for each chromosome

Question 7

In Situ Hybridization pros/cons

Answer 7

Advantages:
Gives cellular localization of specific mRNA and genes being expressed or the number of chromosomes in a cell
Can be used on whole organisms, tissue sections, cells, or chromosomes

Disadvantages:
May be limited by the abundance of the mRNA that is being probed

Question 8

Reverse Transcription – Polymerase Chain ReactionAmplifies DNA or mRNA

Answer 8

Amplifies and detects DNA sequence of interest

Only works with DNA

What if you have mRNA of interest?
First make cDNA from mRNA
then do PCR
mRNA => cDNA => RT-PCR

This method can detect the expression of a specific mRNA (semiquantitative).

Denature DNA with heat, add primer (which binds to the two strands). Replicates until you get as much as you need.

Question 9

Reverse Transcription – Polymerase Chain Reaction pros/cons

Answer 9

Advantages:
Allows for amplification of the DNA/mRNA sample
Few copies of the transcript can be used to amplify and see the signal

Disadvantages:
RT-PCR will not tell the cellular location of the genes being expressed

Question 10

Expression Profiling: DNA Microarray

Answer 10

Can be used to quantify the expression of many genes simultaneously

Similar to RT-PCR except thousands of genes can be studied at one time

Isolate mRNA, label with fluorescent probe. Incubate cell types.

Green is in control, red is experimental yellow is both.

Question 11

DNA Microarray pros/cons

Answer 11

Advantages:
Allows for analysis of multiple genes or the entire genome simultaneously
Tremendous read out of genes expressed

Disadvantages:
Can be Expensive

Question 12

Is mRNA translated?

Answer 12

DNA  RNA  protein

Method usually used to study translation of mRNA into protein is SDS-PAGE followed by Western Blotting

SDS: Sodium dodecyl sulfate
PAGE: Polyacrylamide gel electrophoresis

Question 13

SDS-PAGE Separates Proteins from Each Other

Answer 13

Steps:
Sample of proteins

Proteins are treated with an ionic detergent (SDS) that denatures & unwinds them, and coats them with a negative charge

Mixture is loaded into a gel that separates proteins by size

Run a current through your gel and the proteins will separate by size

Question 14

SDS-PAGE Separates Proteins from Each Other cont.

Answer 14

Now, you have your SDS gel with separated proteins

If you want to visualize the proteins on your gel, you can stain using a coomassie blue stain. (image right)

Question 15

Visualize Specific Proteins using Western Blot

Answer 15

Proteins run on SDS PAGE are transferred to nitrocellulose membrane via current

Question 16

Western Blot

Answer 16

Add primary antibody they will bind to the protein of interest

Question 17

Western Blot cont.

Answer 17

Add secondary antibody bind to the primary antibody.
Secondary antibody must be directed against the species in which the primary antibody was made.

Secondary antibody has a luciferase reporter and cleaves luciferin which generates light and is detected using film (which is the black band)

Presence or absence and the amount of the protein can be determined

Question 18

summary of all Molecular Techniques

Answer 18

DNA or mRNA expression
In Situ Hybridization
Used on embryos, tissues, cells to visualize mRNA expression
Can be used on chromosomes to locate specific genes or determine number of copies of a chromosome

Polymerase Chain Reaction (PCR)
PCR: Amplifies DNA for detection
RT-PCR: Transcribes mRNA into cDNA, then amplifies specific cDNA

Expression profiling: Microarray
Examines expression of multiple genes from a single tissue/cell type simultaneously

Protein expression (mRNA translation)
Western blot
SDS-PAGE: separates proteins according to size
Western Blotting: visualizes protein using antibodies