1. science
2. medicine
3. genetics

Chapter 3
Methods in Molecular Biology
and Genetic Engineering
長庚大學 生物化學科 吳嘉霖 老師 分機 : 5159 The Human Genome Project
2001(draft); 2003(completed)
James D. Watson Before1992
After1992
Celera Genomics
in 1998
The Human Genome Project
strategy
Shotgun sequencing protocols
100,000 genes within 3.2X109 bp
in human genome
Genomic sequencing timeline
Snapshot of the human genome
~ 25,000
PCR and RT-PCR •  RT-PCR uses Reverse Transcriptase to convert RNA to
cDNA for use in a PCR reaction.
•  Real-time, or quantitative, PCR detects the products of
PCR amplification during their synthesis, and is more
sensitive and quantitative than conventional PCR.
PCR and RT-PCR •  fluorescence resonant energy transfer (FRET) – A
process whereby the emission from an excited
fluorophore is captured and reemitted at a longer
wavelength by a nearby second fluorophore whose
excitation spectrum matches the emission frequency of
the first fluorophore.
FIGURE 20: Fluorescence
Resonant Energy Transfer
(FRET)
Fluorescent Tags in Real-­‐Time PCR •  This fluorescent-­‐tagged oligonucleo3de serves as a reporter probe –  Fluorescent tag at 5’-­‐end –  Fluorescence quenching tag at 3’-­‐end •  With PCR rounds the 5’ tag is separated from the 3’ tag •  Fluorescence increases with incorpora3on into DNA product TeqMan probe
1.  Primer binding
2.  Probe hybridization
3.  PCR conditions
wikipedia
SYBR Green I Dye
1.  Primer binding
2.  PCR conditions
SYBR Green I Dye binds to
the minor groove of ds DNA
Reporter dye are not sequence
specific, spurious products
produced by the reaction may lead
to false positive signals.
www.currentprotocols.com
Blotting Methods
•  Southern blotting involves the transfer of DNA from a
gel to a membrane, followed by detection of specific
sequences by hybridization with a labeled probe.
•  Northern blotting, RNA is run on a gel.
•  Western blotting entails separation of proteins on an
SDS gel, transfer to a nitrocellulose membrane, and
detection proteins of interest using antibodies.
FIGURE 21: Southern blot: Identifying Specific DNA Fragments
(Edward Southern--the pioneer)
Gel is soaked in
alkali buffer to
denature DNA
or gentle vacuum pressure
Drying or exposure to UV light
Probes: Isotope or chemical
Northern blotting is similar to Southern blotting, but
involves the transfer of RNA from a gel to a membrane
RNA
How to separate mRNA from all other classes of RNA
How to separate mRNA from all other classes of RNA
mRNA contains ~200 oligo(A)
residues at 3’ end
FIGURE 22: Poly(A)+ RNA can be separated from other RNAs by
fractionation on an oligo(dT) column
Northern blotting: Measuring gene activity
Poly(A)+ RNA: from rat tissues
Probe: G3PDH (glyceraldehyde-3-phosphate dehydrogenase)
Western blotting • Western blotting entails separation of proteins on an SDS
gel, transfer to a nitrocellulose membrane, and detection
proteins of interest using antibodies.
SDS unfold the proteins to make them migrate according to their size, and
also provide uniform negative charge
wikipedia
FIGURE 23: Western blot
Blotting Methods •  Antibodies can recognize the protein of interest or an
epitope tag.
•  epitope tag – A short peptide sequence that encodes a
recognition site (“epitope”) for an antibody, typically
fused to a protein of interest for detection or purification
by the antibody.
Human influenza hemagglutinin (HA): YPYDVPDYA
The HA tag is derived from the HA-molecule corresponding to amino acids
98-106 has been extensively used as a general epitope tag in expression
vectors.
DNA Microarrays •  Gene expression array are used to detect the level of
all the expressed genes in an experimental sample.
•  SNP arrays permit genome-wide genotyping of single
nucleotide polymorphisms. =>use allele-specific
oligonucledtide probe
•  Array comparative genome hybridization (arrayCGH) allows the detection of copy number changes in
any DNA sequence compared between two samples.
Photolithography to create a DNA microarray
The 5’ hydroxyl of nucleotide is blocked,
these blocking groups can be removed by
light DNA Microarrays
•  DNA microarrays comprise
known DNA sequences spotted
or synthesized on a small chip.
FIGURE 24: Microarrays
show the levels of all the
expressed genes in an
experimental sample.
DNA Microarrays
Next-generation pyrosequencing
1.  Addition of nucleotide is detected
with flashes of light
2.  Four deoxynucleotide
triphosphates are pulsed onto the
reacting surface one at time, in a
repeating sequence
3.  Excess nucleotide is destroyed
quickly with enzyme apyrase
before the next nucleotide pulse
4.  Pyrophosphate is released as
byproduct when specific
nucleotide is added to the strand.
5.  Luciferase reacts with luciferin in
the presence of ATP
Next-generation pyrosequencing
Next-generation reversible terminator sequencing
Chromatin Immunoprecipitation(ChIP)
Sonication to
200~1000bp
PCR, Blotting method,
array(ChIP-on-chip)
Chromatin immunoprecipitation (ChIP) allows detection of specific
protein–DNA interactions in vivo.
The yeast two-hybrid systemdefine protein-protein interactions
The yeast two-hybrid
Gene Knockouts and Transgenics
•  transgenics – Organisms created by introducing
DNA prepared in test tubes into the germline.
–  The DNA may be inserted into the genome or exist in
an extrachromosomal structure.
FIGURE 26: Transfected DNA
can be incorporated into the
mouse genome
Photo reproduced from P. Chambon, Sci.
Am. 244 (1981): 60-71. Used with
permission of Pierre Chambon, Institute of
Genetics and Molecular and Cellular
Biology, College of France.
Defective genes can be replaced by
functional genes using transgenic
techniques GnRH (gonadotropin-releasing hormone)
GAP (GnRH-associated peptide)
Gene Knockouts and Transgenics •  ES (embryonic stem) cells that are injected into
a mouse blastocyst generate descendant cells
that become part of a chimeric adult mouse.
–  When the ES cells contribute to the germline, the next
generation of mice may be derived from the ES cell.
–  Genes can be added to the mouse germline by
transfecting them into ES cells before the cells are
added to the blastocyst.
3.12 Gene Knockouts and Transgenics FIGURE 28: ES cells can be used to generate mouse chimeras
The Nobel Prize in Medicine 2007 University of Utah, Salt
Lake City, UT, USA,
Howard Hughes
Medical Institute
Cardiff University,
Cardiff, United
Kingdom
University of North
Carolina at Chapel
Hill, Chapel Hill, NC,
USA
For the discoveries of principles for introducing specific gene modifications in
mice by the use of embryonic stem cells
www.nobelprize.org
Gene Knockouts and Transgenics •  An endogenous gene can be replaced by a transfected
gene using homologous recombination.
•  The occurrence of successful homologous recombination
can be detected by using two selectable markers, one of
which is incorporated with the integrated gene, the other
of which is lost when recombination occurs.
neoR gene: resistant to the drug
G418
TK: Thymidine Kinase (sensitive to
gancyclovir)
TK phosphorylates gancyclovir,
which make it toxic
Homologus recombination
involves two exchanges within
the sequence of donor gene
FIGURE 29: Transgenes can be selected in ES cell
Gene Knockouts and Transgenics Gene Knockout:
Gene deletions
Gene knock-in:
Replacement of a gene with alternative form
Gene Knockdown:
Reduce the amount of gene product
The Cre/lox system is widely used to make inducible
knockouts and knock-ins.
Gene Knockout FIGURE 30: Cre excises the
sequence between lox sites
Structure from Protein Data Bank:
1OUQ. E. Ennifar, et al., Nucleic Acids
Res. 31 (2003): 5449-5460.
FIGURE 31: Cre/lox excises a target only when Cre is activated
Cre/lox system
Cre/lox system
34-base loxP sequence
cre.jax.org
Gene Knock-in FIGURE 32: A knock-in replaces an endogenous gene with an alternative
sequence
knock-in involves a gene inserted into a specific locus, and is a "targeted" insertion
Inducible Gene Expression
and Gene Modification in
Transgenic Mice
tet-OFF
Tetracycline-Inducible
Systems
tet-ON
JAISSER F 2000
Inducible Gene Expression
and Gene Modification in
Transgenic Mice
Cre/lox system and its use
as an inducible expression
system
Cre-ER (ER: Estrogen receptor)
system
JAISSER F 2000
Brainbow
Brainbow
In vitro assay
Livet J et al., 2007
Brainbow
Livet J et al., 2007
In vitro assay
Brainbow
Thy1 promoter
Tamoxifen induce expression
Thy1- Brainbow-1.0 mice line H
crossed with the retina-specific
Chx10-Cre driver.
Livet J et al., 2007
Brainbow
three brainbow transgenes
Livet J et al., 2007
In vivo assay
Brainbow