Biotechnology and Recombinant DNA - Applications

Recombinant DNA (3)

Cut DNA of interest with restriction enzyme

Cut DNA of vector (e.g., plasmid) using the same restriction enzyme - compatible ends

Combine vector and gene of interest using ligase = recombinant

Recombinant DNA - recap and continue (5)

Cut DNA of interest and plasmid vector using the same restriction enzyme - compatible ends

Combine vector and gene of interest using ligase = recombinant

Put recombinant DNA into bacterial host via transformation - transformants are ampR

Plate on amp-containing medium (selective medium)

Grow pure cultures of cells with recombinant plasmid = cloning

Gene Libraries (5)

A collection of bacterial or phage clones

Each contains a portion of the genetic material of interest

Typically, each clone in a library contains one gene of an organism’s genome

A library can contain all of the genes of an organism

A library can contain cDNA’s of an organisms’ expressed genes

Gene Libraries (“Shotgun” Libraries)

Cut DNA of interest with restriction enzyme

Cut DNA of vector (e.g., plasmid) using the same restriction enzyme - compatible ends

Combine vectors and genes of interest using ligase = recombinants

Transform bacteria with mixed recombinants, grow - library

Selecting the Right Clone

Our library contains all of the DNA of the organism = mixture

Must find the clone that contains the DNA of interest
Make replica of master plate (has all of the clones)

How to find clone that contains the DNA of interest (6)

Make replica of master plate (has all of the clones)

Prepare DNA from clones on replica (SDS)

Make DNA on replica single-stranded (NaOH)

Add labeled probes (=single-stranded DNA complementary to gene of interest → hybridize)

Remove unbound probe → expose replica to X-ray film

Compare x-ray film of replica master plate → identify DNA of interest

Inserting DNA into cells

A major goal of recombinant DNA technology is to insert DNA into new cells

Can use vectors and natural methods of transformation, transduction, and conjugation → inefficient and limited

What are artificial methods of inserting DNA into cells?

Electroporation

Protoplast fusion

Lipofection

Injection -gene gun and microinjection

Electroporation and Protoplast fusion

Electroporation = a form of transformation

Electric current creates pores in cell membrane → environmental DNA enters cell

Lipofection = transformation using liposomes (micelles), gentler

Protoplast fusion is used for walled cells

Creates Hybrid Cells

The Gene Gun

Used for walled cells

Uses percussive force to “blast” desired DNA into cells

Microinjection

DNA of interest is injected into the target cell’s nucleus

Used for certain in vitro fertilization techniques (e.g., sperm that can’t penetrate ovum)

Used to produce cloned animals, e.g., Dolly the sheep, the first cloned animal.

Separating DNA molecules: Gel electrophoresis and Southern Blot Analysis

Electrophoresis involves separating molecules based on electrical charge, size, and shape

Gel electrophoresis allows the DNA of interest to be separated, and then identified using Southern Blot analysis

Agarose Gel Electrophoresis

Agarose gel acts as a molecular sieve → large DNA fragments migrate slower than smaller fragments

DNA has a net negative charge due to the sugar-phosphate backbone

Electrical current “drives” the DNA fragments through the gel

Agarose Gel Electrophoresis continued

Ethidium bromide (EtBr) is an intercalating agent and slips between the DNA bases → stains DNA

Stained DNA glows when exposed to UV light

Allows visualization of DNA fragments following gel electrophoresis

Southern Blotting

DNA is transferred from gel to nitrocellulose membrane

Radioactive probes homologous to part of the DNA of interest are used to locate the DNA of interest

Uses of Southern Blot analysis

Genetic “fingerprinting”

Diagnosis of infectious disease

Demonstrate incidence and prevalence of organisms that cannot be cultured

Southern Blotting Cont. (process)

Cut DNA of interest with restriction enzyme → perform gel electrophoresis to separate fragments

Gel is fragile and bulky → transfer DNA fragments to filter (“blot”)

Hybridize with labelled probe → wash off unbound probe

Expose to X-ray film → visualize fragment of interest

DNA fingerprinting

Simple/small genomes (e.g., bacteriophage) yield a small number of discrete fragments when cut → discrete bands on gel after electrophoresis

Complex/large genomes (e.g., human) yield a large number of similar sized fragments when cut → “smear” on gel after electrophoresis.

DNA Fingerprinting Cont.

Human DNA forms a “smear” when subjected to Southern blotting

How can we resolve this so that we can identify people by their DNA?

Use VNTR (variable number tandem repeat) probes

Everyone has a unique pattern = “bar code”

Who committed the crime?

Nucleotide sequencing

Used to determine the order of the bases in a piece of DNA

Used in Human Genome Project

Used for taxonomy (determine the similarity of sequences → determine relatedness/phylogeny)

Use for personalized medicine

Automated Nucleotide Sequencing

No notes in pptx (automated nucleotide sequencing)

Multiplying DNA: The Polymerase Chain Reaction (PCR)

Produces a large number of identical molecules of DNA in vitro

Amplifying DNA is critical in a variety of situations

• Epidemiologists used PCR to amplify the genome of an unknown pathogen (West Nile Virus)

• Use for analyzing ancient DNA

• Used forensically to amplify DNA in blood, semen, tissue samples

Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR)

Repetitive process consisting of three steps

1. Denaturation

2. Priming (annealing)

3. Extension

Can be automated by using a thermocycler

PCR cont. (process)

1. Heat-denature DNA → two (2) single-stranded molecules

2. Add primers + dNTPs + Taq DNA pol

3. Annealing — primers attach to single-stranded DNA

4. Extension — each single-strand is replicated → two copies of DNA = amplification

5. Repeat steps 1-4 to amplify original DNA

• 1→2→4→8→16→32

Norovirus Outbreak (questions)

Are the outbreaks of food-borne illness related?

Is there a common source?

What is the source?

School, publishers, and social workers fed food catered by a restaurant.

Norovirus Outbreak (actually explained)

Norovirus is an RNA virus

Need to make a cDNA

RT-PCR with a Norovirus primer

Generate a unique 213 bp fragment

All of the afflicted show this unique fragment

Common source = sick restaurant worker who washed hands in vegetable prep sink

DNA microarrays

Known DNA (e.g., oncogenes) is immobilized on glass slides or silicon chips

Fluorescently-labeled DNA (probe) is washed over the array

Probe adheres where there is matching DNA

Used to:

• Monitor gene expression

• Diagnose infection

• Genetic screening