Chapter 19

Recombinant DNA Technology

Basic Concepts
The

first recombinant DNA experiments were done in 1973

Stanley

Cohen (Stanford U.) and Herbert Boyer (UCSF) made the first recombinant DNA molecule
DNA from two different sources to create a new DNA molecule

Combined

Recombinant DNA Technology

Techniques

for manipulating DNA

Recombinant

means DNA from different sources is combined to create a novel DNA molecule
Also

use the general term of genetic engineering

Impact of Recombinant DNA

Milestone Prior

in biological studies

to recombinant DNA technology, scientists had to:

Infer

the genotype from the phenotype

Mutations

were limited to inducing random

mutations

Biotechnology
Recombinant

DNA that developed in university research labs naturally led to the development of commercial products
Drugs
Hormones

Genetically

engineered food crops

Working at the Molecular Level

Genetic

studies at the molecular level present formidable challenges do you identify the gene of interest?

How

Suppose

YFG (your favorite gene) is 3,300 bp in the human genome

The

human genome is ~3.3 billion (3.3 X is

th 1/1,000,000 -6) (10

9 10

bp)
YFG

of the genome

Once

you locate YFG, how do you produce enough of the DNA in a form pure enough for biochemical studies? if you want to make the protein encoded by YFG?

What

As

we have discussed, there are significant differences in DNA replication, transcription and translation between bacteria and human cells? expression of a human gene in a bacterial cells presents yet another set of challenges

Proper

Cutting and Joining DNA Fragments

Fundamental

requirement for DNA

manipulation
Possible

by the discovery of a group of bacterial enzymes called restriction endonucleases

Commonly

called restriction enzymes

Restriction Enzymes
Make

double-stranded cuts in DNA

defense against viruses

Bacterial Degrade Why

foreign DNA

isnt the bacterial genome degraded?

Bacterial

DNA is marked by DNA methylation to prevent degradation by its own restriction enzymes

Three Types of Restriction Enzymes

Type

Recognize

a specific DNA sequence, but cut the DNA at a distance ~1,000 bp away useful for recombinant DNA II a specific DNA sequence and cut the DNA within that

Not Type

Recognize

sequence
These

are the enzymes used for molecular cloning

Type Very

III
recently found

Similar

to Type I, but the DNA is cut about 25 bp from the recognition sequence after a number of bacterial genomes had been sequenced

Found

Restriction Enzymes
More

than 800 different enzymes that recognize more than 100 different DNA sequences have been identified enzymes recognize the same DNA sequence most commonly used enzymes are commercially available

Some

The

Common Features of Restriction Enzymes

The

first part of the name identifies the bacterial species from which it was isolated
EcoRI

was isolated from E. coli

Most

recognize a 4 to 8 bp sequence with 4 and 6 bp recognition sequences being most common

Recognition

sequence is always a palindrome for Type II enzymes

Reads 5 3

the same backward and forward

A A G C T T 3 T T C G A A 5

DNA Cleavage
A

phosphodiester bond is cleaved on both strands within the recognition site

cuts

Staggered Creates Called

ends with short single-stranded tails

cohesive ends since they are complementary cuts

Blunt-ended Creates

ends with no single-stranded structure

 HindIII

creates cohesive ends (sticky ends)

PvuII

creates blunt-ends

DNA

from different sources can be digested with the same restriction enzyme to produce complementary termini.

Omit the Following Sections

Viewing

DNA Fragments (pp. 517-18)

Locating

DNA Fragments with Southern Blotting and Probes (pp. 518-19)

Critically

1990s

important technology during the 1980s and early

Now

largely replaced by other methods, especially PCR

Cloning Vectors
A

cloning vector is used as a vehicle to facilitate replication of foreign DNA (also called target DNA)

cloning vector will have

1.An origin of replication that functions within the host cell

2.A selectable marker to select for cells that contain the vector
3.One or more unique restriction enzyme sites to allow insertion of the target DNA

Plasmid Vectors
The

first cloning vectors

Derived

from naturally occurring bacterial plasmids widely used

Still A

diverse array of highly specialized plasmid cloning vectors

Insertion of Target DNA

Target

DNA must be joined to the vector DNA

Usually Other

achieved by using restriction enzymes that create compatible ends methods are available

Leave

the technical details to the biotechnology lab courses

Transformation
Joining

of the target DNA to the vector DNA is done in a test tube DNA must then be transferred to a

Recombinant

host cell
Process Variety

is called transformation

of techniques involving chemical treatment or electroporation

Selective Markers
Cloning

vector needs a selectable marker to select for the host cells that were successfully transformed
Commonly

use antibiotic resistance, e.g. ampicillin resistance

Vector

contains the gene needed for the resistance to the antibiotic

lacZ Gene
lacZ

codes for -galactosidase (used with cloning vectors assay using X-Gal

gal)
Widely Colorimeteric

Cells

expressing -gal turn blue

Plasmid Cloning
Suggest

watching the animation entitled Plasmid Cloning for Chapter 19 on the web site for the textbook (http://bcs.whfreeman.com/pierce4e)

Bacteriophage Vectors
We

have discussed bacteriophage lambda at several times during the course

Developed
Rarely Will

as a cloning vector

used any longer

not discuss further

Expression Vectors
Specially

constructed plasmid vector to allow expression of the protein encoded by the target gene.
all of the properties previously discussed for a plasmid cloning vector

Requires

Also

requires

A bacterial promoter

sequence that codes for a ribosome binding site on the mRNA transcription and termination sequences to control initiation of transcription, regulatory genes and operators (regulatory elements) make the target function as a bacterial gene

Bacterial

Sequences

Must

Cloning Vectors for Eukaryotes

Extensive

array of highly specialized vectors for use with eukaryotic cells

Shuttle

vectors
to work with two different host cells

Designed Most

well developed are yeast shuttle vectors that can be used in either yeast or E. coli cells

Yeast Artificial Chromosome (YAC)

Contains

an origin of replication (ARS), centromere and two telomeres DNA molecules of 1 million bp and even larger use yeast cells as the host cell

Clone

Must

Bacterial Artificial Chromosomes

Lead

to development of artificial chromosomes for use in bacteria to clone fairly large DNA fragments

Used

Up

to ~500,000 bp, which is about 10-fold larger than previously possible using plasmidbased vectors

Polymerase Chain Reaction (PCR)

PCR

is a simple test tube reaction that allows a DNA fragment to be amplified more than one billion-fold in just a few hours watershed technology that opened many new lines of investigation and application most sensitive method for DNA analysis

The

Can

analyze DNA from a single cell

Requirements for PCR

Two

oligonucleotide primers usually 20-30 nt long is a fully automated process

ssDNA,

Synthesis Must

know the sequences at each end of the DNA sequence you want to amplify

Originally,

PCR only worked over fairly short distances, typically no more than 2,000 or so base pairs

Current

technology allows amplification of DNA at least 50,000 bp

The It

most sensitive technique for DNA analysis

is becoming common place to do DNA analysis using PCR on a single cell concept is simple

The

Repeated

rounds of DNA synthesis with the amount of DNA doubling after each cycle 30 cycles, the DNA has been amplified more than one billion fold

After

 Repetitive

rounds of DNA synthesis

DNA

content doubles exponentially, usually for 25-40 cycles

to more than 1,000,000-fold amplification

Leads

Automated PCR
To

make PCR a useful technique, the process needed to be automated DNA to nearly 100C to denature it will also denature most DNA polymerases

Heating

Needed

a heat-stable DNA polymerase

Taq Polymerase
The

first heat stable DNA polymerase was isolated from Thermus aquaticus, a microbe that lives in very hot environments - Taq polymerase does not have proofreading activity
Makes

Problem

an error about once every 20,000 bp

Several

heat-stable DNA polymerases with proofreading activity are now available

PCR

Suggest

watching the animation entitled Polymerase Chain Reaction for Chapter 19 on the web site for the textbook (http://bcs.whfreeman.com/pierce4e)

Real-Time PCR
A

modification of the PCR amplification reaction that measures the amount of PCR product made at the end of each cycle for quantitative measurements of changes in gene expression (i.e. mRNA level) in different cell samples such as normal cells compared to cancer cells Instruments are fairly expensive - probably in the range of $30-$50,000

Valuable

Genomic Libraries
A

genomic library is a collection of clones that, together, represent all of the genomic DNA sequences must be broken down into manageable size fragments clone contains one piece of the genomic DNA

DNA

Each

cDNA Libraries
A

critically important technology

cDNA

stands for complementary DNA (or sometimes called copy DNA) DNA copy of mRNA

Double-stranded

Reverse Transcriptase
Require

a DNA polymerase that uses RNA as a template

First

discovered in retroviruses, such as

HIV
The

key step is the synthesis of a complementary DNA strand using mRNA as a template

The double-stranded cDNA is cloned into a plasmid vector and then used to transform E. coli as described for creating a genomic library.

Screening Libraries
Text

describes several approaches to screen a genomic or cDNA library for your gene of interest.
requires creative thinking and the approach(es) will depend on what information is known about the gene of interest

Often

Some of the methods described are becoming outdated due to genomic sequencing

Look briefly at isolation of the gene responsible for cystic fibrosis

In situ Hybridization
DNA

or RNA probes are used to determine the cellular or chromosomal location of the gene or its gene product previously - FISH (fluorescence in situ hybridization)

Discussed

In Silico Gene Discovery

Gene

discovery is becoming more and more the result of data mining of the extensive and rapidly growing DNA databases new frontier in genetics that is collectively called genomics
terms include bioinformatics, functional genomics, proteomes, transcriptomes will discuss in more detail in the next chapter

Other

We

Cystic Fibrosis (CF)

Autosomal The

recessive trait

CF gene could be anywhere on any of the 22 autosomal chromosomes for a DNA sequence of probably between 5-20,000 bp among the 3.2 billion bp of the human genome

Looking

 The

mRNA for one candidate gene was expressed at high levels in lung, pancreas and sweat glands, all organs affected by CF of CF patients had the 3-bp deletion in this gene

68%

Remaining
Finally,

patients had other mutations within the same gene

showed that the gene coded for a membrane protein involved in chloride transport - cystic fibrosis transmembrane conductance regulator (CFTR) gene.

DNA Fingerprinting
Many

sequences within the human genome are highly variable among different individuals - polymorphic
of these polymorphic sequences are microsatellites or short tandem repeats (see Chapter 11). sequences of interest are amplified by PCR

Many

The

DNA

can come from the individual of interest or a biological source such as hair, tissue, semen, etc.

DNA Sequencing
DNA

sequencing determines the precise nucleotide order in a DNA fragment fully automated process

Now

far cheaper to send it out than do it yourself

Molecular Analysis of Gene Function

Forward genetics

- begin with a known phenotype and then isolate the gene responsible for it - begin with a gene of unknown function and then determine the phenotype when the gene is mutated

Reverse genetics

Random and Site-Specific Mutations

Classical genetics is the forward genetic approach and involves making random mutations in a large number of cells

Then look for the desired phenotype

Reverse genetics requires manipulation of wild-type gene, usually prior to putting the gene back into a cell or organism Techniques are available to change DNA sequences at nucleotide level site-directed mutagenesis

Techniques are sophisticated and beyond the scope of this course

Transgenic Animals
Germline Genetic

transformation

manipulation that leads to well defined genetic changes in cells that form the germline

Progeny

carry the transgene in every cell and pass it on to their offspring

Transgenic Mouse
Just

after fertilization, there are two pronuclei from the gamete of each parent

One DNA A

can be directly injected into one of the pronuclei

few injected eggs will have the transgene inserted at a random site in the genome

Injected

gene is transplanted into a surrogate mother

mouse
A

few transplants are successful and develop into fully developed mice transformants for the transgene

Germline

Another approach involves manipulation of the genome in embryonic stem cells

Knockout Mouse
A

slight twist on the approach of making a transgenic animal knockout the gene of interest to study the effect in vivo

Selectively

The

techniques are sophisticated, but are quite well developed at this time disruption is done in mouse embryonic stem

Gene

cells
Cells

are injected into a mouse embryo that is at a very early stage of development

Gene Silencing
siRNA

is rapidly becoming an important method for knocking down gene expression, especially in mammalian cells
a vector to express the double-stranded RNA needed for gene silencing converts to siRNA through the activity of the Dicer protein

Design

Cell

Read

the section on application of siRNA to treatment of human diseases