Key videos & reading

Griffiths et al., (2015) 10: 351-392

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 DNA purification & amplification

Restriction endonuclease

purification sequence-specific
primers

amplification

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 Restriction enzymes

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 plasmid

Plasmids (red) contain a

gene(s) for antibiotic
resistance, as well as Ori
sequences so they can
replicate in bacteria
(plasmids often referred to
as vectors)

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221.S16
 Cloning vector
A replicating genetic element
used to clone (purify and amplify) a DNA segment
in a living cell
cells vector capacity
E. coli Plasmids 1-10 kb
Bacteriophage 1-20 kb

Cosmids 35-45 kb
Bacteriophage P1 artificial
80-100 kb
chromosome (PAC)

Bacterial artificial chromosome (BAC) 200-300 kb

yeast Yeast artificial chromosome (YAC) 150-500 kb

plasmid
An autonomously replicating
extrachromosomal DNA molecule
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 Plasmid cloning vector

capacity: ~10 kb

oc mutation

lacZ deletion

221.S16 Griffiths et al. (2011) F10-9

 Structure of a BAC vector

capacity: 200-300 kb

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 Turning mRNA into DNA
reverse transcription to make complementary DNA (cDNA)

Single-stranded RNA

RNA-dependent
DNA polymerase

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 Turning mRNA into DNA
reverse transcription to make complementary DNA (cDNA)

RNA/DNA hybrid

DNA-dependent
DNA polymerase

Double-stranded DNA

ds cDNAs can be placed under the control of promoter sequences that drive
their transcription in cells or transgenic organisms

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 Producing cDNA molecules with sticky ends

5
5

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 DNA purification & amplification

Restriction endonuclease

purification sequence-specific
primers

amplification

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 Polymerase chain reaction

Cycle 1

* The critical role of Taq

polymerase (Thermus
aquaticus)

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 Polymerase chain reaction

Cycle 2

Cycle 3

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 Polymerase chain reaction

221.S16 https://www.dnalc.org/resources/animations/pcr.html
 Polymerase chain reaction

221.S16 https://www.dnalc.org/resources/animations/pcr.html
 DNA sequencing: Sanger method

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 DNA polymerization

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 Dideoxy termination of DNA polymerization

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 Dideoxy termination of DNA polymerization

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 Dideoxy termination of DNA polymerization

5 label primer

5
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 Dideoxy termination of DNA polymerization

label ddNTPs

5
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 Automated DNA sequencing (circa 1995)

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 Automated Sanger DNA sequencing (circa 1995)

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 Automated Sanger DNA sequencing (circa 1999)

Capillary electrophoresis (16)

Detector

Sample injection

ABI 3130 Genetic Analyzer

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 Automated Sanger DNA sequencing (circa 1999)

Detector

Interpret

A green
Read
G black
C blue
estimate: 16 lanes (950 bp/lane/3 hr) = 122,000 bp/da T red
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 Automated Sanger DNA sequencing (circa 2004)

Applied Biosystems (ABI) Model 3730xl

estimate: 96 lanes (850 bp/lane/2 hr) = 979,000 bp/da

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 Next Generation Sequencing (2014)
Amplified Single Molecule Sequencing

~10M

Illumina HiSeq x Ten

estimate: 6 x 109 reads/run (2 x 150 bp/read) = 1.2 x 1012 bp/ 3-da

Illumina / ABI = 1.2 x 1012 bp / 3 (~1 x 106) bp = 400,000

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 DNA sequencing costs

Roche 454
sequencing Illumina
sequencing

Moores law: an axiom of microprocessor development usually

holding that processing power doubles about every 18 months
especially relative to cost or size.

http://systems.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/datasheet-hiseq-x-ten.pdf
 Measuring mRNA expression quantitative RT-PCR

5 3 mRNA
AAAAAAA
TTTTTTT DNA
3 5
RT

5 3
AAAAAAA mRNA
TTTTTTT 5 DNA
3

Remove RNA, add primers, Taq pol

DNA
3 TTTTTTT 5
PCR

PCR reaction kinetics are

exponential, not linear!
Quantifying is tricky -
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 Quantitative RT-PCR

Ct: Cycle threshold

Threshold cycle

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Gene construction

cis-acting elements transcription

ds cDNA

promoter
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 Can use reporter genes to identify cis-acting elements in gene
promoters

cis-acting elements YFG

promoter region

YFG reporter gene

typically:

proteinfusion

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 Identification of cis-acting elements using reporter genes
and deletions

cis-acting elements YFG

Expression

YFG reporter gene

100

YFG reporter gene

( ) 1

YFG reporter gene

( ) 100

(aka promoter bashing)

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 Gene construction: Producing PCR products with sticky ends

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 Prokaryotic transcription: factor-independent termination

Bacterial
plasmid

Control Experimental
PBAD: Promoter from Arabinose operon
RBS: Ribosome Binding Site T : Terminator (experimental)
T : Terminator T : Terminator (always present)
GFP: Green Fluorescent Protein
RFP: Red Fluorescent Protein
ColE1: Ori from high copy number plasmid
ampR: Ampicillin Resistance Gene
37
araC: repressor factor-independent (intrinsic)
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 Transfection

Plasmid DNA can be coated with a

cationic polymer (e.g., lipofectamine),
and presented to cells, which will
transport the DNA to the nucleus,
allowing cloned genes to be
expressed

Also modified retroviruses or

lentiviruses can be used to introduce
genes into cells.

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 Transgenic organisms
Reporter gene
A gene whose phenotypic expression is easily monitored.
For example:
-galactosidase (E. coli lacZ) - enzyme activity forming dark blue precipitate
green fluorescent protein (jellyfish GFP) - green fluorescence
luciferase (firefly) enzyme cleaves substrate (luciferin), releasing light

GFP-expressing mice

luciferase-expressing tobacco plant

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