PLNT3140 Introductory Cytogenetics

Lecture 6

September 21,  2017

RECOMBINANT DNA

Genomes 2, T.A. Brown, Chapter 4

GENERAL OBJECTIVES:
Learning checklist
1. DNA structure - know the nucleotides, be able to recognize nitrogenous bases, 5', 3', ends, phosphate-sugar backbone, major and minor grooves; know base-pairing rules; short hand notation for double-stranded and single-stranded nucleic acids
2. Restriction endonucleases  - understand how type II REs work; be able to recognize cutting sites, symmetry, 5' or 3' protruding ends; calculation of expected frequencies of restriction recognition sequences.
3. Principles of separation of DNA molecules by size using gel electrophoresis


I.DNA STRUCTURE
 
DEMO: Space-filling model of DNA

A. Nitrogenous bases (In-class exercise)

B. Nucleosides and Nucleotides (In class exercise)

        Things to remember:

C. DNA and RNA Primary Structure



from http://upload.wikimedia.org/wikipedia/commons/a/a2/AT_DNA_base_pair.png
from http://upload.wikimedia.org/wikipedia/commons/d/d3/GC_DNA_base_pair.png
Note: A:T has 2 hydrogen bonds, weaker; G:C has 3 hydrogen bonds, stronger
A good summary of the structure of DNA can be found at http://youtu.be/ZGHkHMoyC5I

D. Representation of DNA molecules

E. Conventions w.r.t transcription, translation

 

II.RESTRICTION ENDONUCLEASES

A. Definitions

B. TypeII Restriction Endonucleases

A mononucleotide occurs every
4 1=
4 bases

dinucleotide
4 2=
16 bases

tri-
4 3=
64 bases
eg. codon
tetra-
4 4=
256 bases
eg. TaqI
penta-
4 5=
1024 bases
eg. MboII
hexa-
4 6=
4096 bases
eg. HindIII
hepta-
4 7=
16384 bases
eg. AbeI
octa-
4 8=
65536 bases
eg. NotI

For more on restriction endonucleases, see REBASE ( http://rebase.neb.com ).


III. AGAROSE GEL ELECTROPHORESIS

A. Apparatus

Typical agarose gel apparatus

Undisplayed Graphic

Image from NOAA: Deep Sea Medicines
http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate.html

B. Theory


Mobility of a molecule in an electric field is a function of the  charge:mass ratio of the molecule. DNA has a net negative charge at neutral pH due to its phosphate backbone. Thus,  DNA will migrate from cathode (-) to anode (+). DNA is an anion.

D  =  a  -  (b log(M) )   where
D is distance migrated
M is molecular weight or length
a & b are constants


Undisplayed Graphic


BioRad Video on Gel Electrophoresis http://youtu.be/vq759wKCCUQ

C. Separation of molecules based on size

You can include ethidium bromide in the gel, which will intercalate between bases in dsDNA. Under UV light, intercalated EtBr will flouresce a pink color, which can be seen and photographed.


In the example below, DNA from Lambda phage and two synthetic plasmids has been digested with restriction enzymes and electrophoreses, giving  discrete bands .

Lambda phage DNA is less than 50,000 (50kb) long, while the plasmids pAB96.3 and pUC19 are only a few kb.

In contrast, genomic DNA from bacterial genomes is millions of base pairs long, while genomic DNA from eukaryotic chromosomes may be tens of millions to hundreds of millions of base pairs. Consequently, digests of genomic DNA yeild thousands of bands, which can not be resolved individually . Hence, a genomic digest looks like a continuous smear, as shown below. The one exception is high-copy number repetitive sequences which show bands because each copy has the same restriction sites (arrows)

RESTRICTION DIGESTS OF LAMBDA, pUC19 from (WFT)BF45

restriction digests of
                  lambda and pUC19

Rat genomic DNA, digested with HindIII (B) or BamHI (C)
Lane A contains a Lambda-HindIII marker




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