Appendix 1 - Molec Bio techniques

Question 1

ELISA

Answer 1

Enzyme-linked Immuno-Sorbent Assay

antigen-antibody = immuno-sorbent

Assist in determining the presence of:
1) Antiges
2) specific immunoglubulins (antibodies)

Process:
Wells are coated with antibody, specific to an antigen.
Wells are washed
Secondary antibody linked to a detection ENZYME is added that is specific to the antigen.

Often used to detect a virus.

Question 2

Radioimmunoassay

Answer 2

Similar to ELISA, but uses a radiolabeled antigen or antibodies.

More commonly used medically to detect hormone or drug levels.

A standard curve can be developed using a known concentration of radiolabeled antigen with a known concentration of antibody. Additional antigen is added that is not radiolabeled, which creates a standard curve.

This standard curve can be used to compare to a patients blood serum.

Question 2

Electrophoresis

Answer 2

A means to seperate things by size or charge.

A gel is made from acrylamide or agarose. Concentration of this mixture dictates how open the pores of the ‘net’ are.

Wells are loaded with sample. A charge is applied across the gel.

Migrate toward positive pole depending on size and charge.

Can transfer RNA and DNA from gel in a process known as blotting.

Question 3

Blotting

Answer 3

The transfer of RNA or DNA from a gel to a nitroceullose or PVDF membrane.

Probing can then be performed to detect a specific fragment of DNA or Protein.

Categorized by molecule being probed:

Southern blotting: Allows for detection of DNA fragment in a heterogenous sample.

Northern blotting: Same as southern, but RNA is seperated.

Western blotting: proteins

Eastern blots: analyze post-translational modification - though these techniques are not commonly used. Ex. addition of lipids and carbohydrates.

Question 4

Recombinant DNA

Answer 4

A recombinant protein is one that results from obtaining, translating, and transcribing a novel piece of DNA from a different organism.

Often employ the use of restriction endonucleases = bacterial enzymes that recognize and cleave a specific segment of dsDNA. Endo - because it cleaves in the middle.

Question 5

Plasmids

Answer 5

Small circular ds-DNA that are capable of autonomous replication.

Plasmids are usually developed to include a drug resistant sequence to help separate them from non-plasmid containing DNA.

Bacterial expression plasmids also require a prokaryotic promoter, and a start site.

Transient plasmid - remains in the cytosol

Stable transformations - integrated into the DNA of bacteria.

Question 6

Methods of Bacterial Transformation of Plasmids

Answer 6

Bacteria often need to be coaxed to take up DNA.

Methods include:
1) calcium chloride cooled, and then heat shocked

2) electroporation

Question 7

Barriers to replicating Eukaryotic DNA in prokaryotes

Answer 7

Prokaryotes lack the ability to splice out introns.

Eukaryotic DNA is often very long.

Question 8

Methods to express Eukaryotic DNA in Prokaryotes

Answer 8

Complimentary DNA - splice mRNA is used as a template to create complimentary DNA such that it lacks introns.

Accomplished with reverse transcriptase, isolated from retroviruses.

Question 9

Artificial Chromosomes

Answer 9

Plasmids are limited in the size of DNA they can transform into a cell. Artificial chromosomes allow for larger sequences to be added

Bacterial Artificial Chromosomes - 100 - 350 kilobase pairs

Yeast Artificial Chromosomes - 100 - 3000 kilobase pairs

Question 10

Eukaryotic Plasmids

Answer 10

Have additional criteria for successful integration:

Puromycin and neomycin used as selecting agents.

Different promoting agents.

Ploy-A tail.

Question 11

Methods for integrating eukaryotic plasmids into mammalian cells

Answer 11

Chemical transfusion with calcium phosphate precipitates.

Plasmid packaging in lipids.

Electroporation.

Optical transfection with lasers

Shooting DNA coupled to gold nanoparticles into a cell nucleus.

Question 12

Polymerase Chain Reaction

Answer 12

Detection and amplification of specific gene sequences.

Question 13

RT-PCR

Answer 13

Measures relative amounts of mRNA.

cDNA produced from mRNA using reversetranscriptase.

Then undergoes typical PCR with specific primers to determined whether mRNA was expressed at the time.

Question 14

quantitative PCR

Answer 14

or Real-time PCR.

Amplified DNA is detected in real time, can be done with a dye or a fluorescent oligonucleotide probe that hybridizes to a sequence of interest.

Can be performed on cDNA or DNA.

Question 15

Sanger Method

Answer 15

DNA sequencing

Uses modified dNTPs, dideoxynucleotidetriphosphates - when integrated into a chain of nucleotides they prevent further propogation because the 3’ hydroxy group is no longer present.

Four different tubes with the same sequence, but a different NTP (one of the four) are used.

Separate fragments via electrophoresis. Can read sequence based on longest column to shortest.

Question 16

Restriction fragment length polymorphisms

Answer 16

Endonucleases cut 10-100 bp segments out of polymorphic regions of DNA (called mini satelities).

Because of the length variations inherent in these regions, resulting DNA fragments vary in size and can be used to identify individuals.

Note that point mutations, can cause division of fragments to occur differently, so a sequence need only vary by a single nucleotide to have this technique function.

Question 17

Short-tandem repeat analysis

Answer 17

STR analysis.

Uses PCR to amplify 5-10 bp segments of highly polymorphic sections in introns. Will vary with sequence and number of repeats.

Seperated with electrophoresis and then analyzed with southern blotting.

Question 18

Exome and Targeted Sequencing

Answer 18

Full genome sequencing is very expensive.

Exome sequencing - only sequences exons.

Targeted sequencing - targets a specific gene.

Question 19

Fluorescence in situ Hybridization (FISH)

Answer 19

Uses a fluorescent probe of a specific DNA sequence to locate where a specific