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Chapter 3 Methods in Molecular Biology and Genetic Engineering. 3.9 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.

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slide1

Chapter 3

Methods in Molecular Biology and Genetic Engineering

3 9 blotting methods
3.9 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.
slide3

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

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Northern blotting is similar to Southern blotting, but involves the transfer of RNA from a gel to a membrane

RNA

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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

slide7

Northern blotting: Measuring gene activity

Poly(A)+ RNA: from rat tissues

Probe: G3PDH (glyceraldehyde-3-phosphate dehydrogenase)

From Dr. Yu

western blotting
Western blotting
  • Western blotting entails separation of proteins on an SDS gel, transfer to a nitrocellulose membrane, and detection proteins of interest using antibodies.

wikipedia

3 9 blotting methods1
3.9 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.

3 10 dna microarrays
3.10 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 (array-CGH) allows the detection of copy number changes in any DNA sequence compared between two samples.
3 10 dna microarrays1
3.10 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.

3 11 chromatin immunoprecipitation chip
3.11 Chromatin Immunoprecipitation(ChIP)

Sonication to 200~1000bp

Chromatin immunoprecipitation (ChIP) allows detection of specific protein–DNA interactions in vivo.

PCR or Blotting method

3 12 gene knockouts and transgenics
3.12 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.

3 12 gene knockouts and transgenics1
3.12 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.
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Defective genes can be replaced by functional genes using transgenic techniques

GnRH (gonadotropin-releasing hormone)

GAP (GnRH-associated peptide)

the nobel prize in medicine 2007
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

Forthediscoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells

www.nobelprize.org

3 12 gene knockouts and transgenics2
3.12 Gene Knockouts and Transgenics

FIGURE 28: ES cells can be used to generate mice

3 12 gene knockouts and transgenics3
3.12 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.
slide20

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

slide21

3.12 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
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.

slide25

Cre/lox system

34-base loxP sequence

cre.jax.org

slide26

Gene Knock-in

FIGURE 32: A knock-in replaces an endogenous gene with an alternative sequence

slide28

Inducible Gene Expression and Gene Modification in Transgenic Mice

Tetracycline-Inducible Systems

JAISSER F 2000

slide29

Inducible Gene Expression and Gene Modification in Transgenic Mice

Cre/lox system and its use as an inducible expression system

Cre-ER (ER:Estrogenreceptor)system

JAISSER F 2000

slide31

Brainbow

Livet J et al., 2007

slide32

Brainbow

Livet J et al., 2007

slide33

Brainbow

Livet J et al., 2007

slide34

Brainbow

Livet J et al., 2007