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Trends in Biotechnology

150409 TB11 PCR, Sequencing, PAGE,

Microarrays

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Restriction Fragment Length Polymorphism

Video:http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

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Polymerase Chain Reac-tion

Enables researchers to quickly make a spe-cific DNA without building and screening a library.

Has three major steps (called cycles), re-peated about twenty-five to forty times:

1. Denaturing of DNA by high temperatures. 2. Attachment of single-stranded DNA primers

that flank the desired DNA at a lower tem-perature.

3. DNA is synthesized by a special heat-stable DNA polymerase called “Taq DNA polymerase” and isolated from the thermophilic bacterium Thermus aquaticus.

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Fig. 3.22 Polymerase chain reaction is a very good method for multiplying

specific sequences of DNA.

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Short fragments of DNA called oligonu-cleotides act as primers to allow a spe-cific DNA to be replicated by DNA poly-

merase.

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Fig. 3.24 The poly-merase

chain reac-tion (PCR) is an in vitro DNA repli-

cation method.

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Video: 19-polymerase-chain-reactionhttp://www.dnalc.org/files/videos/dnai/mp4/19-polymerase-chain-reaction.mp4

Animation: pcrhttp://www.dnalc.org/resources/animations/pcr.html

Video: http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

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Here is a song.www.youtube.com/watch?

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Sing Along

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PCR is used to: 1. Isolate specific sequences for

further study. 2. Identify specific genetic loci for

diagnostic or medical purposes.3. Generate DNA fingerprints to de-

termine genetic relationships or in forensics.

4. Provide specific DNA for rapid sequencing.

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Real-Time qRT-PCR (Real-Time Quantitative Reverse

Transcription PCR) • reliable detection and measurement

of products made during each cycle of PCR process.

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Use an oligonucleotide probe which hybridizes within the target sequence.

The probe gets cut during PCR because of the 5' nuclease activity of Taq polymerase

This can be used to detect amplification of the target-specific product.

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TaqMan assay (named after Taq DNA polymerase)

Early method Widely adopted for both the quantification

of mRNAs and for detecting variation. Uses the 5' endonuclease activity of Taq

DNA polymerase to cut an oligonucleotide probe during PCR.

Probes fluorescently labeled at their 5' end non-extendable at their 3' end by chemical modification.

Specificity from two PCR primers and the probe.

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Applications of Real Time Quantitative RT-PCR

Relative and absolute quantification of gene expression.

Validation of DNA microarray results.Variation analysis including SNP

discovery and validation.Counting bacterial, viral, or fungal

loads, etc.

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Sanger Sequencing

A DNA primer is annealed to the desired DNA.

DNA polymerase extends the primer, and la-beled nucleotides incorporate in the newly made DNA.

2’, 3’-dideoxynucleotides are incorporated and stop DNA synthesis in four test tubes, with each tube containing one dideoxynucleotide such as ddATP, ddGTP, ddCTP, and ddTTP.

Each resulting strand is a different length, and is separated by electrophoresis.

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Fig. 3.25 The structure of a

dideoxynucleotide shows the substi-tution of an H in-stead of an OH on the 3’ carbon of the deoxyribose.

20Fig. 3.26 DNA sequencing: the “A” tube reaction.

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Fig. 3.27 A Sanger dideoxy DNA sequencing showing (a) the random incorporation of

dideoxy ATP into the growing DNA chain. (b) The radiolabeled products of each reaction

mixture are separated by gel electrophoresis and located by exposure to x-ray film.

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Fig. 3.27 (c) An ex-posed x-ray film showing the se-

quence in all four lanes.

The four lanes repre-sent A, C, G, and T dideoxy reaction

mixes.

23www.kensbiorefs.com/MolecularGen.html

24www.kensbiorefs.com/MolecularGen.html

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Fig. 3.28 Auto-mated DNA se-

quencing.

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Automating Sanger Sequenc-ing

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Video: 29-sanger-sequencinghttp://www.dnalc.org/resources/3d/29-sanger-sequencing.htmlVideo: sangerseqhttp://www.dnalc.org/resources/animations/sangerseq.htmlVideo: cycseqhttp://www.dnalc.org/resources/anima-

tions/cycseq.html

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A. DNA sequencing lets us: 1.Confirm the identity of genes isolated from

DNA libraries. 2.Find the DNA sequence of regulatory DNA

elements such as promoters.3.Reveal the fine structure of genes and

other DNA.4.Confirm the DNA sequence of cDNA and

other DNA made in the laboratory.5.Determine the amino acid sequence of a

gene or cDNA from the DNA sequence.

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Protein Methods

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Protein Gel Electrophoresis • used to separate individual

proteins by size or charge.• one-dimensional elec-

trophoresis uses polyacry-lamide gel (PAGE).

• gives bands of protein.

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Fig. 3.29 The five histone proteins

that asso-ciate with eukaryotic DNA are shown.

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Two-dimensional electrophoresis has two steps.

a) Isoelectric focusing separates proteins in a pH gradient. The pro-teins move until they have no charge. This is called the “isoelectric point.”

b) This gel is then put on another gel which has sodium dodecyl sulfate (SDS-PAGE). SDS gives proteins a negative charge. The electrophoresis separates the protein by size.

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The Iso-Electric Focusing (IEF) separates pro-teins on the basis of their charges. The SDS PAGE separates proteins on the basis of their size.

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Fig. 3.30 Hu-man HeLa cell proteins sepa-rated by two-dimensional

gel elec-trophoresis are seen as spots.

Henrietta Lacks

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Video Protein 2D Electrophoresis:http://highered.mheducation.com/sites/9834092339/student_view0/chapter20/electrophoresis.html

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Protein engineering changes the protein’s gene sequence to change the amino acid se-quence, and so change the pro-tein’s properties. It may make the protein function better or worse.

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DNA Microarray Technology uses hybridization.

It can be used to: 1. Analyze gene activity, ie

gene expression (transcription). 2. Follow changes in genomic

DNA.

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Each spot on the slide or chip represents the location of many copies of a signal DNA sequence.

39Fig. 3.34 The steps in preparing a microarray, preparing and hybridizing la-beled target cDNA, and analyzing the data.

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Video 26-microarray:http://www.dnalc.org/resources/3d/26-microarray.html

Transcript:The grid you can see in this microarray slide is ac-

tually composed of 30,000 individual DNA dots, each targeted to match a specific human gene. In order to work out which genes are being ex-pressed, messenger RNA is first extracted from the cell sample and copied back to DNA using an enzyme. This DNA, called cDNA, is complemen-tary to the target gene, so we'll associate with it, or hybridize with it, on the slide. After labeling with a fluorescent dye, the cDNA is washed over the slide. The genes currently active in the cell can then be identified by the level of the fluores-cence and the color of the spots.

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Microarrays: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro50.swf::MicroarrayDnaarray:http://www.dnalc.org/resources/animations/dnaarray.htmlDnachip:http://www.dnalc.org/resources/animations/dnachip.html

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http://www.bio.davidson.edu/Courses/genomics/chip/chip.htmlhttp://www.dbc.uci.edu/~faculty/wagner/array2.htmlhttp://www.biostudio.com/d_%20Synthesizing%20Oligonucleotide%20Microarray.htm

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Animation 36: Different genes are active in different kinds of cells. Igor Dawid & Thomas Sargent. interested in "differentiation" genes. subtractive mRNA hybridization to find the genes expressed by different cell types. looking at the differences in the mRNA population between different developmental

stages of frogs. EG, the blastula is a ball of undifferentiated cells, whereas the ectoderm, endoderm, and

mesoderm cell layers develop in the gastrula stage. Isolated mRNA from the different stages. made DNA complements — cDNA — of the mRNA collected from the gastrula stage. mixed the gastrula cDNA with blastula mRNA; the mRNA found in both stages will end up

as a cDNA-mRNA hybrid pair. hydroxyapatite column binds the double-stranded cDNA-mRNA hybrids. Ie subtracted the

mRNAs common to both stages, left with a population of cDNAs unique to the gastrula stage. separated these cDNAs and then inserted them into plasmids and clone them.a library of clones of gastrula-specific cDNAs . use the unique cDNAs as radioactive probes on developmental dot blots. spotted mRNAs collected from the different stages and times of development onto nitro-

cellulose. incubate the strips with the radioactive cDNAs, which hybridize to mRNA spotted onto

the strips. Strips give a visual record of the stages and times of development of these genes. In situ hybridization, (in situ is Latin for "in state") shows where mRNA is expressed

within an organism. .

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Pat Brown. developed a technique where cDNAs can be embedded onto glass slides. - DNA arrays, large-scale expression studies. unique cDNA populations are directly embedded onto specially-coated glass slides

coated with poly-lysine, (positively charged. )Use arrays to get gene expression profiles of cancer. Diffuse large B-cell lymphoma (DLBCL) Sub-types of DLBCL correlate with survival rates. A chip with genes expressed by the lymph nodes and those important in cancer biology. 17,856 cDNA genes printed onto the "lymphochip." Make cDNAs from different DLBCL tumors. Label one set of cDNA with a red fluorescent tag; the other with a green tag. Incubated the arrays with the tagged cDNAs, bind to the matching genesThe positions of the genes on the DNA array, and the levels of gene expression based on

the color signal. If the gene was only expressed in DLBCL1 cells, the square was red, and, if the gene was

only expressed in DLBCL2 cells, the square was green. If the gene was expressed equally in both cells, the square was yellow.

Two sub-types of DLBCL — GC B-like and Activated B-like DLBCL. These sub-types have different responses to therapy, and with this type of diagnosis,

more targeted treatment can begin for patients. This type of targeted treatment is called "pharmacogenomics." Analyze the differences in gene expression for these two very similar lymphomas. → bet-

ter understanding of how cancers work → develop better therapies and cures.

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Hi, I'm Stephen Fodor. GeneChip® probe arrays - build the sequences DNA sequences are built up using light-directed chemical synthesis. Substrate with a nucleotide is fixed onto the chip at specific positions. The nucleotide has a protecting group (X) that blocks polymerization. This protector group is photolabile and is released on exposure to UV light. Without the protector, polymerization and chain build-up occur. Add a filter to the chip so that only some of the nucleotides are exposed to light. These deprotected groups are then free to add the next nucleotide to the chain. By alternating the position of the filter, we can build a GeneChip® with an array of different sequences about 20 nucleotides long. Simultaneously assay tens of thousands of different sequences at the same time. With a computer, pinpoint the matching sequences, which can then be matched to avail-able DNA sequences in gene databases. Eg. BRCA-2, which has been implicated in causing human breast and ovarian cancer. Include sequences of known BRCA-2 mutations, → a diagnostic test for women at risk for these cancers. Single nucleotide polymorphisms (SNPs), can be the ultimate DNA fingerprint. Genetic disease mutations can be imprinted and used for diagnosis or more tailored drug therapies