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The Principle and Application of Common Used Techniques in Molecular Biology

chapter 18

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1

Molecular Hybridization and

Blotting Technique

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nucleic acid hybridization

In DNA renaturation process, if the

type of single-stranded DNA molecule in

the same solution, the DNA or RNA

together with, as long as between single-

stranded DNA or RNA molecule with a

base pairing relationship can between the

different double-stranded hybrid

molecules

1.Principles and blot hybridization techniques

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Refolding

RNA DNA

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BlotVarious physical methods using gel

electrophoretic transfer to NC biological

macromolecules other film, making the

solid phase molecule. This technique is

similar to absorb the ink on the paper with

a blotter, so called "blotting", translated

blot technique.

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Radionuclide, biotin or a fluorescent

dye labeled polynucleotide chain end or

the whole chain of known sequence is

called a "probe", the probe may bind to

the immobilized polynucleotide of the NC

membrane, determine whether

homologous nucleic acid molecule.

Probe Technology

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2. Category Blot and application

(1) Southern blotting

（ 2 ） Northern blotting

（ 3 ） Western blotting

Analysis of genomic DNA, recombinant plasmids and bacteriophages used.

For qualitative and quantitative analysis of RNA.

Quantitative and qualitative for protein interaction studies.

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Other ： dot blotting

in situ hybridization

DNA array

DNA chip

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Com

paris

on

of th

ree

kin

ds o

f Blo

t

Molecular hybridization experiments

①

②

③

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Autoradiograph

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DNA chip

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2

The Principle and Application of

PCR Technology

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5 Primer 15 Primer 2

Cycle 2

Cycle 1

55

5

5

5 5

Template DNA

1 、 Principles of PCR technology

55

5

55

5

5 5

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Cycle 3

5

5

5

55

5

5 55

5 5 5

5 55

5

After 25 to 30 cycles, the content of the template DNA can be expanded 100 times or more.

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Schematic diagram of PCR technology

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The basic steps of PCR reaction

变性95˚C

延伸72˚C

退火Tm-5˚C

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Template DNA

Primers

Thermostable DNA

polymerase

dNTPs

Mg2 +

The basic components of PCR system

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Real-time PCR technology principle

QR

3'

3'

5'

5'Upstream primer

Downstream primer

Fluorescently labeled primers

R

Q

3'

3'

5'

5'

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Real-time PCR TaqMan probe Schematic

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3

Gene Library

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• genomic DNA library

• cDNA library

gene library

Refers to an organism containing a

DNA sequence of the entire population of

clones.

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1. Genomic DNA Library

Genomic DNA library of genomic DNA

is the information on an organism

(including all coding and non-coding

region) in the form of storage of cloning a

DNA fragment populations.Vector used to construct genomic

libraries have phages, cosmids and

yeast artificial chromosomes.

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Construction and screening of genomic libraries and cDNA library

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1 2 3

Examples of genomic library screening results

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cDNA library mRNA that contains a

clonal population of all cells under

certain conditions the expressed cDNA

synthesized by reverse transcription of

the sequence, it is stored in the form of

a cDNA fragment of the gene expression

of cells of information.

2. cDNA library

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4

Biological Chip Technique

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DNA 微阵列 (DNA microarray) 。

1. gene chip

gene chip

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基因芯片工作流程示意图

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The protein molecules are highly

dense arrangement of the lattice as a

probe immobilized on a solid phase

support material, when reacted with the

target protein sample, the sample may

be captured in the target protein, and

then the target protein detection system

for qualitative and quantitative analysis

a technique.

2. Protein chips

蛋白质芯片 (protein chip)