DNA-Based

Technologies
BI OCHE MI STRY I I
DR . CHR I ST IANE HE N N ESSEE
MAR 29, 2016

A. Introduction

Todays Agenda:
My FAVORITE
topics Thinking
of getting this
chapter tattooed
on my arm

B. Cloning techniques
C.

Forensics and fingerprinting

D. Site-directed mutagenesis
E.

Transgenic animals (Gene therapy)

F.

Summary

Have you covered

many of these topics
in other classes?
In good detail?

Recombinant DNA technology used to

better characterize genes and proteins
The fundamental guiding principle of this
chapter is:
How can science take one gene (or
perhaps a few) and understand its
function?
Or conversely, how can they pinpoint the
root genetic cause of a phenotype?
If gene function is understood, can
transgenic therapy be used to treat human
disease?

Genetic manipulations (or recombinant DNA

technologies) allow scientists to answer those
questions.
Expression of a mutant gene to understand
alterations in phenotype
Isolation of a gene and expression in a new
organism/system
Tagging a gene to visually be able to follow it in a
cell, during development, under diverse conditions
Purification of a protein for use in vitro assays
Using genetic libraries to search for interacting
proteins
Genome wide expression studies under various
conditions
Introduction of functional genes via delivery
systems to replace missing functions

Cloning schematic overview

Definition?
Separation of a gene from a chromosome, then amplification or
making multiple copies in a new host

Steps involved?

Cleaving cloning vector (plasmid)

Cleaving gene of interest from chromosome
Ligation
Introduction into a new host (possibly a bacterial cell)
Propagation, producing many copies of both plasmid and host cell

Use of restriction enzymes

What is a restriction endonuclease?
What are sticky versus blunt ends?
What is depicted in the figure here?

How might they be similar to an antibiotic? (Why did

these evolve?)
How do researchers make use of them besides cloning?
In RFLP?
In forensics?

There are hundreds of known REs

REs differ in their
recognition site, so
they can be useful
either by virtue of
recognition sequence
frequency or location.

They might be used in

combination.
Plasmids used for
cloning always have
engineered RE sites.

PCR can be useful to

generate sites at ends
of a DNA fragment for
cloning.

Anatomy of a plasmid
What is a plasmids role in cloning?
Can you explain the features of this plasmid, their
usefulness?
How does a plasmid get inside its new host (say E. coli)
after we are done engineering it with the gene of
interest?
Electroporation and transformation

Cloning with a plasmid vector

Bacterial Artificial Chromosomes

they just hold a lot more data
BACs can hold 100 300 kB,
so they occur in lower copy
number per cell
May be used to house the
whole genome of an
organism ask me how!
What is lacZ?
What is chloramphenicol?

The purpose of cloning might be to

isolate the protein of interest
Examples might include:
Protein will be studied in vitro
Protein has commercial value (Its intended for mass production)
You are interested in the effects of specific mutations on the
function of the protein

Use of expression vector

Plasmid will be of a special type In addition to the other features
of a plasmid, it should have a strong promoter and regulatory
sequences

Terminal tags for purification

Ultimately, you want a way to separate the wheat from the chaff
A fusion protein (target + tag) is created with high affinity to a
ligand
Attaching nucleotides encoding His residues will allow for
chromatographic separation using a column with nickel ion beads

A forensic application of
restriction enzymes
Recall that REs cut at recognition sites
Some sites occur frequently within a genome Result?
What is the consequence of polymorphism between genomes?
RFLP = Restriction Fragment Length Polymorphism
This method relies on
having a large DNA
sample (>25 ng), not
usually available from a
crime scene. To give you
some perspective, that
amount could come from
a lentil-size amount of
bacteria.

So how can the problem of amount or

quality be solved?
Like many other questions, by PCR!
Short tandem repeats (STR) in human genome

Sequences of 4 bases or so repeated at a locus

Number of repeats vary in individuals
Flanking sequences are identical
If amplified by PCR, length will vary
In a sample from a single individual, how many
bands would arise?

Combined DNA Index System

Based on the combination of loci on last slide
The process
PCR amplification from a sample of very little or even
degraded (old) DNA
Primers target regions flanking STR sequences from 16
loci
Using multiplex PCR What is that?
Product subject to capillary electrophoresis
With colored dyes on primers and detector system on
CE, comparison of samples is quick

Chance of accidental match between two individuals

in human population is 1 in 1018
If odds of error in science are so low, why is DNA
science questioned at trials sometimes?
What other applications of this technique exist?

Two methods of site-directed mutagenesis

First method is quite simple:
Relies on having RE cutting
sites at appropriate
distances from desired
nucleotide change
Cut with RE
How do you separate the
two cut pieces?
A synthetic short piece of
DNA that contains both
restriction sites and single
base change
Ligation
Cloning process

Second method is available when RE sites

are not convenient
Denature a plasmid-containing WT gene to
single stranded
Create a synthetic SS short DNA fragment
with mutation, allow to anneal
Polymerase finishes the circle to give
double-stranded plasmid
Cloning process
Within the bacterium, mismatch repair
systems will recognize the incorrect pair.
Half the time, it will remove and replace the
altered base; other half, it will remove the
WT and retain mutant base pair.
Sequencing of clones will reveal which
bacterial clones contain mutant of interest

Two methods of site-directed mutagenesis

First method is quite simple:
Relies on having RE cutting
sites at appropriate
distances from desired
nucleotide change
Cut with RE
How do you separate the
two cut pieces?
A synthetic short piece of
DNA that contains both
restriction sites and single
base change
Ligation
Cloning process

Second method is available when RE sites

are not convenient
Denature a plasmid-containing WT gene to
single stranded
Create a synthetic SS short DNA fragment
with mutation, allow to anneal
Polymerase finishes the circle to give
double-stranded plasmid
Cloning process
Within the bacterium, mismatch repair
systems will recognize the incorrect pair.
Half the time, it will remove and replace the
altered base; other half, it will remove the
WT and retain mutant base pair.
Sequencing of clones will reveal which
bacterial clones contain mutant of interest

What about transgenic animals?

How does the introduction of foreign genes to animals differ than to
yeast or bacteria?
In BIG ways.
Since animals are multicellular, any introduction of novel traits must occur in
the egg or else it is partial or chimeric
Methods of introduction:
There is nothing like a plasmid system that can allow the uptake of DNA from
environment for animal cells
DNA is introduced via microinjection directly into the nucleus of fertilized egg (change
over whole life of organism, establishes a transgenic line of, e.g., mice)
Liposomes (may or may not reach its target)
Viral vectors (effective on the level of introducing new DNA to a multicellular large
organism)

Viral vectors a great idea,

in theory
Animal viruses have evolved over time to be effective delivery
systems of foreign genes, right?
They can attach to host, inject DNA
Some can even integrate DNA into host genome
With modifications to remove genes required for viral replication
and assembly, recombinant viruses can just deliver foreign DNA to a
target cell

So whats the down side?

Where does a virus usually integrate its genome? (A.K.A, what time is it
when an elephant sits on your fence?)
Diploidy can be an issue if the bad copy of the gene
Integration site can even cause risk of cancer say activation of a gene
stimulating cell division

So what is the current and future state of

idea of gene therapy is to deliver, by viral vector
gene therapy? The
or by liposome, DNA that would correct deficiencies
What kinds of diseases might be
treated?
Ethicists established some
guidelines
Well-characterized single-gene
disorder (excluding things like..)
Functional gene must function
well in presence of mutant
(what does that mean?)
Risks of disease must be
greater than those of
procedure

in individuals with diseases of known genetic origin

What kinds of diseases have
been treated?
SCID (Severe Combined Immune
Deficiency )
Problem with immune cell
differentiation arising from
single gene defect in cell
surface cytokine receptor
Bone marrow transplant or live
in sterility
*That chance of cancer? Yup.
Leukemia successfully treated
with chemo

What kinds of diseases

might be treated one
day?
Cancer itself- deliver
genes specifically to
control cell cycle
HIV delivery of DNA
encoding antisense RNA
or a dysfunctional viral
protein subunit

Recombinant DNA technology is a fancy term for mixing and

matching, or cutting and pasting DNA.

Summary of
Todays Lecture

There can be many reasons for doing that.

Cloning is one of the big reasons with many downstream
applications.
Putting one gene into E. coli to mass produce it
Putting one gene into E. coli to study it better.
Putting one DNA into E. coli so you can create mutations that you will use later
inside its real host.

Putting a whole genome inside an easy-to-work-with bacterial host.

Putting a replacement gene into a host to therapeutically supply a missing
phenotype.

Aside from cloning, we can be cutting up DNA for forensic

analysis, or typing of any sort (parental, strains, etc.)

http://proteopedia.org/wiki/index.php/User:Michael
_Patrick/Sandbox_2
The diversity of genetic mutations is illustrated by
this San Diego beach scene drawn with living
bacteria expressing 8 different colors of fluorescent
proteins.

Text pages to read:

pp. see Blackboard

Reading and
Review

Important concepts to learn:

Prerequisite knowledge for this course: PCR (pages 317-318). You must know this,
but I will not cover in class.
Recombinant DNA technology examples, definition
Action of restriction enzymes (exactly which bond they cleave), staggered versus
blunt cuts, concept of recognition sequences, result of digestion of chromosomal DNA
(many fragments)
Features of a plasmid
Cloning definition, procedure (understand and know order)
Selection of positive clones
RFLP, application in forensics for large DNA samples
STR, how used with PCR in CODIS for forensic analysis
Site-directed mutagenesis, two variations, depending on convenience on restriction
sites flanking mutation site
Gene therapy (DNA entry into mammalian cells, methods and challenges); target
diseases for gene therapy; ongoing challenges