Lecture 6

September 20, 2018

RECOMBINANT DNA

REFERENCE

GENERAL OBJECTIVES:

To obtain a working knowledge of recombinant DNA technology, which underlies many of the experiments we will look at in this course.
To build a conceptual model of chromosomes from the level of the nucleotide to the level of the chromatid.
To understand how eukaryotic genomes function in the greater context of the eukaryotic cell.

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

A. Nitrogenous bases (In-class exercise)

B. Nucleosides and Nucleotides (In class exercise)

Things to remember:

nucleoside = base + sugar
ribonucleoside = base + ribose
deoxyribonucleoside = base + 2'-deoxyribose

eg. deoxyAdenosine, deoxyGuanosine, deoxyCytidine, Thymidine (~deoxyThymidine)

alpha and beta phosphates
backbone gives net negative charge
nucleotide = nucleoside 5'-phosphate
ribonucleotide = ribonucleoside 5'-phosphate eg. Adenosine monophosphate (AMP), Adenosine diphosphate (ADP), Adenosine triphosphate (ATP), Guanosine triphosphate (GTP), Cytidine triphosphate (CTP), Uridine triphosphate (UTP).
deoxyribonucleotide = deoxyribonucleoside 5'-phosphate eg. deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP).

C. DNA and RNA Primary Structure

1. The Phosphate-Sugar Backbone (In class exercise)

2. Base Pairing


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

In contrast, RNA structure can be

ss or ds
can form 2° structure

D. Representation of DNA molecules

Equivalent representations:

5'   pGpCpApGpTpApT-OH 3'
3' OH-CpGpTpCpApTpAp   5'

5' GCAGTAT 3'
3' CGTCATA 5'

`5' ------- 3' 3' ------- 5'`

E. Conventions w.r.t transcription, translation

transcription is 5'->3', translation N->C
by convention, 5' end of gene indicates start of transcription, 3' end is end of transcription
euk. mRNA's have 5' caps (pGppp)
5'UTR = 5' untranslated region; 3' UTR = 3' untranslated region; CDS = protein coding sequence

II.RESTRICTION ENDONUCLEASES

A. Definitions

endonuclease - cleaves within strand

eg. Ribonuclease A, forms 3'phosphate after pyrimidine

pUpApCpCpApUpG-OH ---> pUpApCp + HO-CpApUpG-OH

exonuclease - digests from end of strand

eg. E. coli ExonucleaseIII, a 3'->5' exonuclease

pCCCTGACT.....       pCCCTGACT....
 GGGACTGA..... -->     GGACTGA....    + dGMP

B. TypeII Restriction Endonucleases

1. Recognition and cutting sites

(abbreviations from first letter of genus and first two letters of species, possibly a strain designation, and chromatographic fraction number)

EcoRI G^AATTC 5' protruding ends( Escherichia coli)

HindIII A^AGCTT " " " (Haemophilus influenza)

SmaI CCC^GGG blunt ends ( Serratia marcescens)

XmaI C^CCGGG 5' protruding ends (Xanthomonas malvacaerum)
an isoschizomer¹of Sma1

PstI CTGCA^G 3' protruding ends (Providencia stuarti)

HinfI G^ANTC 5' protruding ends (H. influenza).
Degenerate recognition site.
(GAATC,GAGTC,GACTC,GATTC)

HaeII RGCGC^Y 3' protruding ( H. aegyptius )
2²=4 possible cuting sites:
AGCGCC, AGCGCT, GGCGCC, GGCGCT

BglI 5'GCCN NNN^NGGC3'
3'CGGN^NNN NCCG 5' assymetric, 3'protuding, (Bacillus globigii)

BbvI 5'GCAGC(N)₈3'
3'CGTCG(N)₁₂3' assymetric, 3'recessed

¹isoschizomer - restriction endonucleases that recognize the same sequences

R = purine; Y = pyrimidine; N = {A,G,C or T}

DEMO: redemo.obj

2.Frequencies of restriction sites

A mononucleotide occurs every
4¹=

4 bases

dinucleotide
4²=

16 bases

tri-
4³=

64 bases

eg. codon

tetra-
4⁴=

256 bases

eg. TaqI

penta-
4⁵=

1024 bases

eg. MboII

hexa-
4⁶=

4096 bases

eg. HindIII

hepta-
4⁷=

16384 bases

eg. AbeI

octa-
4⁸=

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

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

from Keller S https://www.alpfmedical.info/microsatellite-instability/electrophoresis-of-restrictiondigested.html
Creative Commons Attribution 3.0 license

Unless otherwise cited or referenced, all content on this page is licensed under the Creative Commons License Attribution Share-Alike 2.5 Canada

EcoRI	G^AATTC	5' protruding ends( Escherichia coli)
HindIII	A^AGCTT	" " " (Haemophilus influenza)
SmaI	CCC^GGG	blunt ends ( Serratia marcescens)
XmaI	C^CCGGG	5' protruding ends (Xanthomonas malvacaerum) an isoschizomer¹of Sma1
PstI	CTGCA^G	3' protruding ends (Providencia stuarti)
HinfI	G^ANTC	5' protruding ends (H. influenza). Degenerate recognition site. (GAATC,GAGTC,GACTC,GATTC)
HaeII	RGCGC^Y	3' protruding ( H. aegyptius ) 2²=4 possible cuting sites: AGCGCC, AGCGCT, GGCGCC, GGCGCT
BglI	5'GCCN NNN^NGGC3' 3'CGGN^NNN NCCG 5'	assymetric, 3'protuding, (Bacillus globigii)
BbvI	5'GCAGC(N)₈3' 3'CGTCG(N)₁₂3'	assymetric, 3'recessed
¹isoschizomer - restriction endonucleases that recognize the same sequences R = purine; Y = pyrimidine; N = {A,G,C or T}

A mononucleotide occurs every	4¹=	4 bases
dinucleotide	4²=	16 bases
tri-	4³=	64 bases	eg. codon
tetra-	4⁴=	256 bases	eg. TaqI
penta-	4⁵=	1024 bases	eg. MboII
hexa-	4⁶=	4096 bases	eg. HindIII
hepta-	4⁷=	16384 bases	eg. AbeI
octa-	4⁸=	65536 bases	eg. NotI