CHMI 4226-W20051 Recombinant DNA Technology CHMI 4226 E Week # 4 January 26, 2009 cDNA libraries

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Recombinant DNA TechnologyCHMI 4226 E

Week # 4

January 26, 2009

cDNA libraries

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Genomes, Genes and Organisms

• The genome contains all the genetic information necessary for the formation of a complete, functional living organism;

• Generally, the more evolved an organism, the bigger the genome, and the greater the number of genes.

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Genomes, Genes and Organisms• However, a few

questions arise upon closer examination:

– D. melanogaster (the fruit fly), C. elegans (nematode), A. thaliana (mustard plant) and humans have similar number of genes in their genomes;

– The same set of genes is sufficient for the generation of a wide diversity of organs and tissues (e.g. brain vs liver vs skin).

SpeciesSize of genome

Number of genes

Human

2.9 billion base pairs

20,00-25,000

Fruit fly (Drosophila melanogaster)

120 million base pairs

13,601

Baker's yeast (Saccharomyces cerevisiae)


6, 275

Worm (Caenorhabditis elegans)


19,000

E. coli

4.1 million base pairs

4,800

Arabidopsis (Arabidopsis thaliana)


25,000

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• These observations can be explained by the following:

– Differences in gene expression (both qualitative and quantitative);

– A substantial fraction of genes in higher vertebrates can code for multiple mRNA species through alternative splicing, giving rise to a great variety of protein products.

– Alternative splicing is differentially regulated in different cell types.

http://www.exonhit.com/alternativesplicing/pages/diagrams/figure2.htm

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• Thus, in order to understand the molecular basis of function (proliferation, differentiation, death), one must know which genes are expressed in which cell/tissue type.

• This is often done through the use of cDNA libraries.

• cDNA (complementary DNA): a DNA copy of an mRNA (thus, contains the coding info of the genome – so, NO introns).

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• cDNA libraries: – A mixture of cDNAs each cloned in a vector (usually

a phage-based vector);

– Each cDNA library is prepared from RNA isolated from a specific tissue/organ, and therefore represents the mRNA population (qualitatively and quantitatively) of that cell/tissue at the time of RNA extraction;

– Because cDNA libraries contain thousands of different cDNAs, strategies must be designed to isolate the cDNA of interest (library screening).

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cDNA libraries – basic steps

Cell/Tissue

RNA isolation

cDNA synthesis

Reverse transcription

Clone cDNA in vector

Library screening

Characterize clones

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RNA isolation

Cell/Tissue

Lysis in acid Guanidium thiocyanate Buffer (chaotropic agent – very good at denaturing RNAses)

Acid Phenol extraction-Solubilizes proteins and DNA-RNA stays in GT buffer

Ethanol precipitation

RNA analysis on agarose/formaldehyde gel Total RNA is: - 85% ribosomal RNA

- 15 % transfer RNA - 1-3% mRNA

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Poly (A+) RNA isolation

mRNA’s 3’ end is extended after transcription by a tail of polyadenosine (poly A tail). No other type of RNA has that kind of modification.

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Poly (A+) RNA isolation

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cDNA synthesisRadioactive dCTP is added as tracer.

RNAse H cleaves only the RNA in an RNA/DNA duplex.RNA fragments left by RNAse H act as primers for the synthesis of the coding strand of the cDNA.

EcoR I methylase add CH3 groups to GAATTC sites and prevent EcoR I from cutting the cDNA.

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cDNA synthesis

Size fractionation is necessary in order to ensure that most of the cDNAs in the library are large enough to contain a complete mRNA.

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Phage lambda

Lysogenic cycle

Lytic cycle

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Phage lambda

Clear lysis plaques of phage lambda grown on a lawn of E. coli bacteria

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Phage lambda

gt10 gt11

Genes for phage insertion and excision

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cDNA libraries

WITH helper phage = pBluescriptNO helper phage = lytic cycle

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cDNA libraries

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Screening

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Screening

• Radioactive DNA/RNA probes:– Partial (incomplete) cDNA;– cDNA from a similar molecule from same species;– cDNA from another species;– Oligonucleotide (deduced from amino acid sequence);

• Antibodies;• Functional screening (according to the

properties of the protein).

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cDNA labeling

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DNA hybridization

• Several factors influence the rate of DNA hybridization– Nucleotide sequence

• Relative abondance of AT vs GC

• Length • Presence of repetitive

sequences• Extent of sequence

complementarity (mismatches)

– Temperature– Salt concentration– pH– Additives (e.g. formamide)

95oC

Hybridization (e.g. Tm-5oC)

Detection by autoradiography or phosphorimaging

Wash-out unhybridized probe

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Detection of a radioactive signalAutoradiography

• The X-ray film is a photographic emulsions consisting of silver halide crystals in suspension in a clear gelatinous phase.

• β-particle or a γ-ray emitted by a radionuclide convert Ag+ ions to Ag atoms, which leave a dark precipitate.

• Variables affecting the darkness of the spots include:

– Radioactivity of the sample– Time of exposure– Temperature:

• E.g.: exposing film with 32P-labeled sample at -80oC instead of room temperature increases the signal several fold.

Increasing amounts of radioactivity in sample

Signal saturates rapidly

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Detection of a radioactive signalAutoradiography

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Detection of a radioactive signalStorage Phosphor systems

BaFBr:Eu2+ + - BaFBr:Eu3+

32P/35S emitters

BaFBr:Eu3+

↓h (633nm)BaFBr:Eu2+

+h (390 nm)

Ref: R. Switzer and L. Garrity. Experimental Biochemistry – Theory and exercises in fundamental methods, 1999, 3rd ed. W.H . Freeman and Co.

http://radiographics.rsnajnls.org/content/vol27/issue3/images/large/g07ma02g03x.jpeg

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Detection of a radioactive signalStorage Phosphor systems

http://las.perkinelmer.com/ProductCatalogTrack/Templates/EnlargeImgPopUp.aspx?ImagePath=%2fContent%2fImages%2flargeImages%2fC431220.jpg&AltText=c431220

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Detection of a radioactive signalStorage Phosphor vs autoradiography

Pg A.3A.8

• Advantages of SP vs Autorad:– Dynamic range:

• >105 fold for PS• 10 fold for autorad

– Exposure times 10 to 250 shorter

– Screens are re-usable indefinitely

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Detection by autoradiography

Clones!!!

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Other probes: Expressed Sequence Tags

mRNA isolation cDNA mixtureReverseTranscriptase

Clone cDNAs into plasmids

Sequence millions of cDNA clones

Upload data in EST database

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Other probes: Expressed Sequence Tags• Anonymous cDNA sequences

• Obtained from the random, large scale sequencing of cDNA clones

• Very useful:– Qualitative and quantitative representation of the mRNAs expressed in a

particular cell line (the transcriptome);– Obtain data on the transcriptome of a specific organism under specific

conditions (develpment, tissue-specific expression, stress-related, pathology-related);

– Data on alternative forms of an mRNA (results of alternative splicing)– Makes Blasts searches easier: a hit on an EST will give you the

nucleotide sequence of your cDNA!!

• However: Mostly limited to model organisms (human, rat, mouse, fruit fly, nematode, A. thaliana, S. cerevisiae, S. pombe, a few bacterial species).

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Other probes: Riboprobes

For riboprobe synthesis, an RNA version of the DNA probe is made by in-vitro transcription in the presence of NTPs, an RNA polymerase (T7 or T3 in this case), the DNA fragment of interest, and a radioactive nucleotide (e.g. 32P-ATP).

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Other probes: Oligonucleotides• THESE ARE NOT

PRIMERS!!!!!!!!!!!!!!!!!!!

• The oligonucleotides can then be labeled with T4 DNA kinase in the presence of 32P-ATP

• When designing an oligonucleotide from an amino acid sequence, remember:– The genetic code is

degenerate;– Some codons are more

frequently used than others;

– Keep the degeneracy to a minimum.

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Codon usage in human

http://www.kazusa.or.jp/codon/

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Screeening with

antibodies

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Expression Screening

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Screening via cell panning

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cDNA clones!

Ori C

AmpR

Sequencing

Restriction mapping

Sub-cloning

IdentificationExpressed sequence tag

(EST)

mRNA expressionMutagenesis

Complete?

Blast

probe

5’/3’ RACE

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5’ and 3’ Rapid Amplification of cDNA Ends (RACE)

5’ RACE 3’ RACE

Documents

CHMI 4226-W20051 Recombinant DNA Technology CHMI 4226 E Week # 4 January 26, 2009 cDNA libraries