Lecture21_RecDNA_2015

HOW WAS

THE TEST?

A. Too hard

B. A little too hard

C. Good

D. A little too easy

E. Too easy

BIO215, Spring 2015; Marco Gallio

A. B. C. D. E.

39%

33%

0%

3%

24%

BIO215, Spring 2015; Marco Gallio Great work!


More Lac Operon

what is this recombinant dna you speak of

WHAT ARE THESE THINGS?

A. Bacterial wheels

B. Chloroplasts

C. Holes

D. Plasmids (DNA)

E. Plasmids (RNA)

BIO

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A. B. C. D. E.

4% 4% 2%

90%

0%

these


Isolate, study, manipulate genes from any organism

Key to modern molecular genetics, medicine, biotechnology

Genetic

engineering

USES

Isolate genes to better study the proteins they encode, understand their function in the cell

Isolate and study genes that are causing human diseases - screen drugs, design rational

therapies

Manipulate genes to improve crops, produce useful proteins (insulin), create useful bacteria

for bioremediation


DEFINITIONS:

Recombinant DNA

Recombinant DNA molecules are formed by laboratory methods of genetic recombination to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms.

Molecular Cloning

is a set of experimental methods used to direct the replication of recombinant DNA molecules within host organisms [bacteria, yeast]. cloning refers to the fact that the method involves the replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing identical DNA molecules


NOT THE SAME!

OUR GENETIC ENGINEERING

PROJECT


GOAL

Make fluorescent fish!


+

STEP1:

Isolate the green fluorescence gene = encodes the Green Fluorescent Protein GFP


Replicate the GFP gene in bacteria to study it

= cloning


How do we get the GFP gene?

STEP1:

RESTRICTION ENZYMES CUT DNA AT SPECIFIC SEQUENCES


EcoRI

Structure of the homodimeric

restriction enzyme EcoRI (cyan

and green cartoon diagram)

bound to double stranded DNA

(brown tubes). Two catalytic

magnesium ions (one from each

monomer) are shown as

magenta spheres and are

adjacent to the cleaved sites in

the DNA made by the enzyme

(depicted as gaps in the DNA

backbone)

DNA

EcoRI dimer

1.1 SYSTEMATICALLY CUT THE DNA

1.1 SYSTEMATICALLY CUT THE DNA


One of the more common

RESTRICTION

ENZYMES

RESTRICTION

ENDONUCLEASES


NotI is also a common

RESTRICTION ENZYME

A MULTIPLE CLONING CASSETTE (MCS) CONTAINS MULTIPLE RE SITES

NotI site

With a bacterial promoter for expression


Clone EcoRI fragments Here Which

components

do we need

in a plasmid

vector?

1.2 INSERT THE DNA INTO A VECTOR

CLONING SCHEMATIC


insert

vector


1.3 TRANSFORM BACTERIA


CLONING=

Each colony contains

Identical bacterial

descending

From1 cell = clones.

If transformed each will

have the same plasmid

Genomic library (many plates like this)

WOULD THE JELLYFISH

PROMOTER WORK IN

BACTERIA?

A. Yes

B. NO

C. Dunno


A. B. C.

38%

5%

57%

WE NEED A VECTOR

With a bacterial promoter for expression


Clone cDNAs Here Which

components

do we need

in a

plasmid?

WOULD BACTERIA KNOW

HOW TO SPLICE GFP?

A. Yes

B. No

C. GFP must have

no introns


Yes

No

GFP

mus

t hav

e no

intro

ns

2% 0%

98%


Pre-mRNA

REVERSE TRANSCRIPTION POLYMERASE CHAIN

REACTION

(RT-PCR)


Complementary DNA is an

artificial DNA copy

of each RNA

STAGE 1

STAGE 2

Reverse

transcriptase

DNA

Polymerase


cDNA Library

WHAT IF WE KNEW THE

JELLYFISH GENOME

SEQUENCE?


STEP1:

Discover the green fluorescence gene = encodes the Green Fluorescent Protein GFP


Here we will

assume we know

the genome

sequence of the

jellyfish

MICROARRAY A DNA microarray (also commonly

known as DNA chip) is a collection of

microscopic DNA spots attached to a

solid surface

Each spot (25bp) is

seeded with a known

sequence that

corresponds to a gene

BIO215, Spring

2014; Marco Gallio

tentacles hood


Gene prediction in the genome

GFP SEQUENCE



REAL TIME or quantitative PCR

You can follow the

amplification curve in real

time: quantification of

initial amount of mRNA is

much more accurate

cycle number

We could use this to

confirm expression

BIO

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5,

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(WHAT IF WE DO NOT FIND THE RIGHT SITES

FLANKING THE GFP GENE?)


GFP coding

We can add them to the

primers when we amplify the

GFP from jellyfish by RT-

PCR: theyll get incorporated into the PCR product


Electrophoresis in an agarose gel

CLONING SCHEMATIC


RT-PCR insert

vector

GFP STRUCTURE


SEQUENCING


QUESTIONS?


Lecture21_RecDNA_2015

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