目录 the principle and application of common used techniques in molecular biology chapter 18
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目录
The Principle and Application of Common Used Techniques in Molecular Biology
chapter 18
目录
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)