Molecular biology application in food

biotechnology Part 2. Brief story of Molecular Cloning and its application

Pramono, H.

Outline

• Molecular cloning

– DNA extraction

– Electrophoresis

– PCR

– Cloning

– Gene expression

• Example

DNA extraction

• DNA isolation consist of whole genome and gene isolation

• Whole genome isolation is first step to isolate gene from organism/cells

• DNA which are negative charged always wrapped by cell membrane and some times other partition (cell wall/ peptidoglycan)

• Principles: – disruption of cell membrane and wall

– separate the genome from other substance

– Isolate genomic DNA

Basic Protocol

• Most DNA extraction protocols consist of two parts

1. A technique to lyse the cells gently and solubilize the DNA

2. Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules

• In plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA from the rest of the cell.

1. Lysis

2. Precipitation

3. Wash

4. Resuspension

A comparison of DNA extraction methods used

in research labs as opposed to classroom labs

Research

Lysis: grind in Liquid N2 and use detergent

Precipitation Part I: phenol/chloroform extraction to get rid of proteins

Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA

Precipitation Part III: addition of ethanol to pull DNA out of solution

Wash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.

Classroom

Lysis: grind in mortar/pestel and use detergent

Precipitation Part I: NONE (chemical are too dangerous!)

Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA

Precipitation Part III: addition of ethanol to pull DNA out of solution

Wash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.

Break down

the cell wall

and

membranes

Centrifuge to

separate the

solids from

the dissolved

DNA

Precipitate

the DNA

using

isopropanol

Centrifuge to

separate the

DNA from

the dissolved

salts and

sugarsWash the

DNA pellet

with Ethanol

and dry the

pellet

Dissolve

DNA

Overview of DNA Extraction

1 kbp and 100 bp

laddersGenomic DNA of 5

species of cereals

Expected Results in a Research Lab

Below is an agarose gel that has 5 genomic DNA samples from various plants.

Note that the DNA runs at a very high molecular weight and as a clear, thick band.

This DNA was extracted in a research lab under optimal conditions

Gel Electrophoresis

• Electrophoresis is method to separate

substance (commonly DNA or Protein)

employing agarose or PAGE

• Negative charged molecule will move to

positive part of electrophoresis chamber

• Usually adding Etidium bromide (EtBr) or

another substance that interfere in DNA for

visualization purposes

waynesword.palomar.edu

UV light visualization

enfo.agt.bme.hu

PCR

• Polymerase chain reaction (PCR) is a

method to replicate DNA

• Polymerase (remember replication) from

Thermus aquaticus (Taq polymerase)

commonly uses to elongate process since

this enzyme still firm in ±95oC

• Invented by Karri Mullis

www.slideshare.net

Principles of PCR

www.soi.wide.ad.jp

Molecular cloning

1. Cut the gene

2. Paste the gene on vectors

3. Transform into E.coli

4. Screening process

5. Purification

Gene Cloning

molecularhub.blogspot.com

Cloning Vector

• Cloning vector can be:

– Bacteriophage

– Plasmid

– Cosmid

– Other

Bacteriophage

Cosmid

Plasmid

Lodge et al.

Expression

• Little bit different with cloning

• Using different vector, called expression

vector

• Expression vector can be:

– E. coli

– S. cereviceae

– B. subtilis

• Need induction

www.biomics.cn

Review

• https://www.youtube.com/watch?v=sjwNtQYLKeU

Application of technology

1. Detection of pathogenic bacteria in food

sample

2. Isolating important gene and express its

protein in mass production

3. Bioreactor of some specific and useful

protein

4. Screening of bioactive-producing isolates

Cloning

Detection

Protein Profiling

Home work (group of 5)

• Please make short review (max 12 p) included:

– Basic molecular biology of the cell

– DNA extraction, PCR, gene Cloning, cloning vectors, gene expression, gel electrophoresis, PAGE

– Application of molecular technique in food/fishery technology (minimum explain 5 journal from Sciencedirect, springerlink, or DOAJ)

• Rule:

– A4, Arial 11, margin 4333,

– Consist of Introduction, summary of theory, application of theory (5 journal)

Any question?