Molecular biology （ Chapter 10 ） -2

李 芬

2014年 11月

The Outline of Molecular

Biotechnology

   

Main Content

Genetic engineering and molecular cloning1

DNA amplification2

Gene function identification techniques5

66

Molecular hybridization3

DNA sequencing4

Interactions between biological macromolecules

Main Content

Main Content

DNA sequencing

Automatic DNA sequencing

Maxam-Gilbert Sequencing

Dideoxy chain-termination method

1

2

3

DNA sequencing Dideoxy chain-termination method

Concept ： Dideoxy chain-termination method is a

method of DNA sequencing based on the selective

incorporation of chain-terminating dideoxynucleotides

by DNA polymerase during in vitro DNA replication.

DNA sequencing Maxam–Gilbert sequencing

This method is based on nucleobase-specific partial

chemical modification of DNA and subsequent cleavage

of the DNA backbone at sites adjacent to the modified

nucleotides.

DNA sequencing Automatic DNA sequencing

Introduction ： Automatic DNA sequencing take some of the

drudgery out of DNA sequencing and speed up data

production , The instrument is a microchemical robot that

can carry out the fragmentations of 16 dna solutions-that is it

can test for the four bases(A,G,C and T) in each of four

different dna solutions. it performs the fragmentation in four

to five hours, following the Maxam-Gilbert protocol.

Gene function identification techniques

Transgenic technologyI

Gene knockoutII

Gene SilencingIII

Topic I. Transgenic technology

Transgenic animals

Transgenic plants

Introduction1

2

3

Applications 4

Benefits and Disadvantage5

Concept ： Transgenesis is the process that

introducing an exogenous gene — called a

transgene — into a living organism so that the

organism will exhibit a new property and

transmit that property to its offspring.

Topic1. Transgenic technology

Introduction

Topic1. Transgenic technology

Introduction

Transgenic plants

Topic1. Transgenic technology

1. What are transgenic plants?

2. Methods for producing transgenic plants.

3. The flow chart of producing transgenic plants.

What are transgenic plants?

Transgenic plants are plants that have been

genetically engineered, a breeding approach that

uses recombinant DNA techniques to create plants

with new characteristics.

Transgenic plants

Methods for producing transgenic

plants

VS

Transgenic plants

Methods for producing transgenic

plants

1. Transgenic technology

The flow chart of producing transgenic

plants

Transgenic plants

Transgenic animals

Topic I. Transgenic technology

1. What are transgenic animals?

2. Methods for producing transgenic animals.

3. The flow chart of producing transgenic mice.

Transgenic animals

What are transgenic animals?

Transgenic animals are genetically modified organisms which are animals. They have had their DNA altered specifically by having the DNA of another animal inserted into their own code.

Methods for producing transgenic

animals

Transgenic plants

1. Microinjection （显微注射法）

2. Retroviral infection embryo （逆转录病毒感染胚胎法）

3. Embryonic stem cell method （胚胎干细胞法）

4. Sperm carrier method （精子载体法）

5. Nuclear transplantation method（细胞核移植法）

6. Germ cell transfection method （生殖细胞转染法）

Transgenic mice expressed GFP

Applications

转基因抗性植株筛选

A 抗生素筛选抗性愈伤组织 B 抗性愈伤组织分化培养 C 遗传转化筛选后得到的试管苗

Topic1. Transgenic technology

Food

Topic1. Transgenic technology

Benefits and Disadvantage

Benefits

1. Reduce the production cost.

2. Improve the output.

3. To reduce the harmful material.

4. Add some beneficial to human

body.

Disadvantage

Topic II. Gene knockout

Method

Concept1

2

Use3

Gene knockout

A gene knockout is a genetic technique in which

one of an organism‘s genes are made inoperative.

Concept

Gene knockoutMethod

Knockout mice / 基因敲除小鼠

Gene ofselectivemarker

Target geneon a plasmid

Transform mouse embryonic stem cells

Black mouse

Collectembryonicstem cells

Knockout mice / 基因敲除小鼠

Intact targetgene on thechromosome

Transform mouse embryonic stem cells

Homologousrecombination

Target genedisrupted onone of thechromosomes

ES cell

Knockout mice / 基因敲除小鼠

White mouse

Take earlyembryos

Inject transformedstem cells intoearly embryos

Implant intopseudopreg-nant mouse

Male chimeragrows up

Femalewhitemouse

Understand the role of a gene or DNA region by comparing the knockout organism to a wildtype with a similar geneticbackground.

Development of drugs, to target specific biological processes or deficiencies .

understand the mechanism of action of a drug by using a library of knockout organisms spanning the entire genome, such as in Saccharomyces cerevisiae.

Gene knockoutUSE

Topic III. Gene Silencing

Method

Concept1

2

Use3

Gene Silencing

Gene silencing is a general term used to describe

the epigenetic regulation of gene expression. In

particular, this term refers to the ability of a cell to

prevent the expression of a certain gene.

Concept

1 、 Antisense oligonucleotides2 、 Ribozymes

General mechanism utilized by ribozymes to cleave RNA molecules

Method

Gene Silencing

3 、 RNA interference

RNA interference

siRNA

CellmRNAs

siRNA binds tocomplementary mRNA

Target mRNA is degraded

The gene knockdown / 基因敲低

What happens if it is removed or

altered?

如果它被移去或改变会怎样？

Use

Gene Silencing

Interactions between biological macromolecules

11

22

Interaction between DNA and Protein Interaction between DNA and Protein

Interaction between Protein and Protein Interaction between Protein and Protein

Interaction between DNA and Protein

Gel Shift assay

DNase I footprinting

chromatin immunoprecipitation

ChIP-chip/ChIP-seq

yeast one-hybrid screening system.

1

2

3

4

5

Interaction between DNA and Protein

Gel Shift assay

Concept ： Gel Shift assay (Electrophoretic Mobility

Shift Assay )is capable of binding to a given DNA or

RNA sequence, and can sometimes indicate if more

than one protein molecule is involved in the binding

complex.

Interaction between DNA and Protein

DNase I footprinting

Concept ： A DNase footprinting assay is a DNA

footprinting technique from molecular biology/

biochemistry that detects DNA-protein interaction

using the fact that a protein bound to DNA will often

protect that DNA from enzymatic cleavage.

Concept ： Chromatin Immunoprecipitation (ChIP)

is a type of immunoprecipitation experimental

technique used to investigate the interaction between

proteins and DNA in the cell.

Interaction between DNA and Protein

Chromatin immunoprecipitation

Interaction between DNA and Protein

ChIP-chip/ChIP-seq

Concept ： ChIP-seq is a method used to analyze

protein interactions with DNA.

Concept ： The yeast one-hybrid screening system

is a versatile and efficient method to identify

transcription factors that can bind and regulate a

given gene-of-interest.

Interaction between DNA and Protein

Yeast one-hybrid screening system

Yeast two-hybrid assay 11

Co- Immunoprecipitation 22

GST Pull-Down 33

FRET44

Phage display 55

BiFC 66

Interaction between Protein and Protein

Interaction between Protein and Protein

protein chip77

Interaction between Protein and Protein

Yeast two-hybrid assay

Concept ： Yeast two-hybrid system is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.

Principle ： Co-IP works by selecting an antibody

that targets a known protein that is believed to be a

member of a larger complex of proteins. By

targeting this known member with an antibody it

may become possible to pull the entire protein

complex out of solution and thereby identify

unknown members of the complex.

Interaction between Protein and Protein

Co- Immunoprecipitation

Concept ： GST can be added to a protein of interest

to purify it from solution in a process known as a pull-

down assay.

Principle ： This is accomplished by inserting the GST

DNA coding sequence next to that which codes for the

protein of interest.

Interaction between Protein and Protein

GST Pull-Down

Interaction between Protein and Protein

FRET

Fluorescence resonance energy transfer (FRET) is a mechanism describing energy transfer between two light-sensitive molecules A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.

Concept ： Phage display is a laboratory technique

for the study of protein–protein, protein–peptide,

and protein–DNA interactions that uses

bacteriophages to connect proteins with the genetic

information that encodes them.

Interaction between Protein and Protein

Phage display

The sequence of events that are followed in phage display screening to identify polypeptides that bind with high affinity to desired target protein or DNA sequence .

Interaction between Protein and Protein

BiFC

Concept ： Bimolecular fluorescence

complementation (BiFC) is based on the association

of fluorescent protein fragments that are attached to

components of the same macromolecular complex.

Proteins that are postulated to interact are fused to

unfolded complementary fragments of a fluorescent

reporter protein and expressed in live cells.

Protein complex formation using BiFC. Interaction between protein A and protein B occurs first, followed by the re-formation and fluorescence of fluorescent reporter protein

N fragment C fragment

BiFC

Protein A Protein B

linker

A&B interaction

N fragment C fragment

BiFC

Concept ： A protein chip is a high-throughput

method used to track the interactions and activities of

proteins, and to determine their function, and

determining function on a large scale.

Interaction between Protein and Protein

Protein chip

Gene chip

Introduction ： A DNA chip is a collection of microscopic

DNA spots attached to a solid surface. Scientists use

DNA microarrays to measure the expression levels of

large numbers of genes simultaneously or to genotype

multiple regions of a genome. Each DNA spot contains

picomoles (10−12 moles) of a specific DNA sequence,

known as probes.

Principle ： The core principle behind microarrays is

hybridization between two DNA strands, the

property of complementary nucleic acid sequences to

specifically pair with each other by forming

hydrogen bonds between complementary nucleotide

base pairs.

Gene chip

Hybridization of the target to the probe Hybridization of the target to the probe