Lecture 15 - DNA Manipulation: PCR and Molecular Cloning

Two Laboratory Techniques

Polymerase Chain Reaction (PCR):

• technique that uses the principles of DNA replication

Polymerase Chain Reaction, PCR

• makes use of the principles of DNA replication

• rapidly makes millions of copies of a specific DNA sequence in a few hours by a chain amplification process

• The cycle is repeated 25-40 times

• Each round of amplification doubles the number of molecules that have the same sequence as the template duplex (Cycle n product: 2ⁿ)

PCR Step 1

Denaturation:

• Introduction

  A solution containing double-stranded template DNA is heated to separate the DNA into two individual strands

PCR Step 2

Annealing:

• The solution is cooled, and the two primers anneal to their complementary sequence on the DNA template strands

PCR Step 3

Extension:

• DNA polymerase uses dNTPs to synthesize new DNA strands by extending the primers

• dNTPS: a mix of dATP, dCTP, dGTP, dTTP

Key to PCR: heat stable DNA polymerase

• allows separation of double-stranded DNA by heat (w/o killing the enzyme) alternating with repeated cycles of DNA replication

What if the template is RNA

• DNA polymerase can only use DNA as a template

• First, we need an enzyme that can use RNA as template and make a DNA copy —> Reverse Transcriptase

• DNA copy is called complementary DNA (cDNA)

Plasmids

• found naturally in bacteria

• are small, circular molecules of DNA distinct from the bacterial chromosome

• can be used to generate more copies of our gene of interest

Plasmids in the lab

Circular double stranded DNA with three essential components:

1. an origin of replication (Ori)

2. an antibiotic resistance gene (e.g. ampicillin resistance)

3. a cloning site to insert DNA of interest

Steps of Molecular Cloning

1. DNA segment of interest is inserted into the plasmid at the multiple cloning site

2. Transform the recombinant plasmid into bacteria

3. “Grow” more plasmid

Restriction Enzymes

• endonucleases that cut DNA at specific sequences. There are hundreds of restriction enzymes, each cuts at a different sequence

• The recognition site is often a palindrome

Gel Electrophoresis

• separates DNA fragments by size. The DNA fragment of interest can be isolated from the other cut fragments using gel electrophoresis

Ligation

• the plasmid and the DNA fragment of interest are cut with the same restriction enzyme to generate complementary sticky ends

• DNA Ligase then seal the backbone

Transformation

• genetic alteration in a cell as a result of uptake and expression of foreign genetic material

• technique for efficient transformation is to increase permeability of bacterial cell membrane by chemical treatment or electrical shock

Procedure for Selection

• plate transformed bacteria on a plate

• select for selectable marker on plasmid using antibiotics

• plasmid containing bacterial cells will grow to form “colonies”