RECOMBINANT DNA

Introduction

• Recombinant DNA

– A gene which has been constructed in the lab

using two pieces of DNA from different biological

sources

– The technology used to create and study the

molecules described

Cloning

• Insertion of a gene or other DNA sequence

into a bacterial cell, which will then be used

as a factory to produce millions of copies of

the gene of interest

Cloning

• Steps in cloning:

– Gene of interest is purified from cells or tissues

– Restriction enzymes cut DNA

– DNA fragments are ligated to a vector, such as a plasmid

– Vector is transferred to a host cell

– Host cell multiplies naturally into millions of cells, each

containing copies of the DNA

– Cloned DNA is purified from the host cells

OR…

– Gene product (protein) can be purified from host cells

Examples of Restriction Enzymes

Vectors

• Acceptors and carriers of DNA to be cloned

• Characteristics of vectors

– Contain several restriction sites

– Must be introduced into a host cell for replication of

vector

– Should have a selectable marker gene

– May contain nucleotide sequences that allow for

sequencing the inserted DNA

• Vectors are usually modified bacterial plasmids

Example of Plasmid

How do we know the cells took up the plasmid?

How do we know the plasmid contained the gene

of interest?

Example of Recombinant Plasmid

Replicating Inserted DNA

Clicker Question: How do you know

whether a host has taken up a vector

during a typical cloning experiment?

• A) It produces a blue colony

• B) It produces a white colony

• C) It produces any colony

• D) It fails to grow at all

Clicker Question: What do you use to cut

a plasmid?

• A) An exonuclease

• B) A restriction endonuclease

• C) Tiny scissors

In Vitro DNA Amplification:

Polymerase Chain Reaction (PCR) • PCR accelerated DNA duplication

compared with cloning

• Reaction components:

– Taq DNA polymerase

– Mg2+ ions

– Deoxyribonucleotide triphosphates (A,T,C,G)

– Primers specific for the DNA to be amplified

– Template DNA

– Optional: Reporter dye

In Vitro DNA Amplification: PCR

PCR Analysis

Reverse-Transcriptase (RT)-PCR

• Start with mRNA

– Isolate from tissue, cells, blood, plants,

bacteria, etc.

• Use reverse transcriptase to create cDNA

– RT: viral enzyme

– cDNA: complementary DNA

• Amplify cDNA using PCR

• Use to determine gene expression

After PCR… (possibilities)

• Run the product (amplicon) on a gel

• Compare CT values between groups

• Purify the product

• Sequence the product

• Insert the product into a vector

Clicker Question: What is special about

Taq polymerase?

• A) It is especially fast at polymerizing

• B) It can proofread better than other DNA

polymerases

• C) It can bend the space-time continuum

• D) It can withstand high temperatures

Clicker Question: How does Dr. DuPriest

use PCR in her research?

• A) To create new vectors

• B) To determine size of fragments

• C) To compare gene expression between

groups

• D) To compare samples between a

suspect and a crime scene specimen

Restriction Mapping

Uses of Restriction Mapping

• Mapping a segment of DNA

• Chromosomal analysis

• Genetic testing (markers)

• Forensic analysis – comparing DNA of

different individuals

Blotting

Types of Blotting

• Southern blotting

– Measures presence / amount of specific DNA

in a sample

• Northern blotting

– …specific RNA in a sample

• Western blotting

– …specific protein in a sample

• All use specific probes to detect specific

molecules (DNA/RNA/antibody)

DNA Sequencing

DNA Sequencing

DNA Sequencing

Clicker Question: Which type of

blotting has largely been

replaced by PCR?

• A) Southern

• B) Northern

• C) Western

• D) Eastern

Clicker Question: DNA

sequencing has largely

replaced…

• A) Western blotting

• B) Cloning

• C) Restriction mapping

• D) PCR