Biotechnology and Genetic EngineeringPBIO 4500/5500

•Characterization of DNA clones including:•Restriction Enzyme (RE) mapping

•Subcloning•Southerns•Northerns*•Westerns

•Hybrid-select translation*•DNA sequencing*

•PCR

Characterization: RE mapping

Predict what would happen with a double digest.

Characterization: Subcloning

• Refers to the process of cloning smaller pieces of a large DNA cloning into another cloning vector

• E.g., subcloning the individual EcoRI fragments of a partial EcoRI lambda genomic clone into plasmid vectors

• Facilitates amplication and analysis of the subcloned DNA, including probe preparation

Characterization: Southern blot hybridization

-transfer of DNA from a gel to a membrane (e.g., nitrocellulose, nylon)-developed by Edwin Southern

Characterization: Northern blot hybridization

-transfer of RNA from a gel to a membrane (e.g., nitrocellulose, nylon)-reveals mRNA size (and approximate protein size), tissue- and organ-specific expression, and kinetic patterns of expression

X RNA

X

xsalt

XRNA

Copyright © 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.

Experimental Figure 5.27 Northern blot analysis reveals increased expression of -globin mRNA in

differentiated erythroleukemia cells.

Copyright © 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.

Experimental Figure 5.28 In situ hybridization can detect activity of specific genes in whole and

sectioned embryos.

Characterization: Western blotting

-transfer of protein from a gel to a membrane (e.g., nitrocellulose, nylon)-requires the use of an electric current to facilitate transfer

X Protein

X

x Buffer; requires electric current

X

React withAntibody

X

Enzymereaction

or

Animations for Western Blotting

• SDS gel electrophoresis- MCB Chapter 3• http://bcs.whfreeman.com/lodish7e/#800911__812035__

• Western blotting or Immunoblotting- MCB Chapter 3• http://bcs.whfreeman.com/lodish7e/#800911__812034__

Characterization: Hybrid-select

translation

Characterization: DNA Sequencing

The dideoxynucleotide structure (note 3’H) is the key to dideoxy DNA sequencing

Dideoxy DNA Sequencing Animation- MCB Chapter 5

• http://bcs.whfreeman.com/lodish7e/#800911__812048__

Characterization: DNA sequencing

DNA sequencing

AutomatedDNA sequencing

Capillary electrophoresis

Step 1A sequencing primer is hybridized to a single stranded, PCR amplified, DNA template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate (APS) and luciferin.

Step 2The first of four deoxynucleotide triphosphates (dNTP) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide.

(DNA)n +dNTP DNA Polymerase> (DNA)n+1 + PPi

Step 3ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram™. Each light signal is proportional to the number of nucleotides incorporated.

Step 4Apyrase, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added.

Step 5Addition of dNTPs is performed one at a time. It should be noted that deoxyadenosine alfa-thio triphosphate (dATPS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase.As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peak in the Pyrogram® trace.

Pyrosequencing http://www.biotagebio.com/DynPage.aspx?id=7454

454 Sequencing (massively parallel pyrosequencing)How it WorksSystem Workflow: One Fragment = One Bead = One ReadThe complete sequencing workflow of the Genome Sequencer FLX System comprises four main steps, leading from purified DNA to

analyzed results. These basic steps include: 1) Generation of a single-stranded template DNA library, 2)Emulsion-based clonal amplification of the library, 3) Data generation via sequencing-by-synthesis, and 4) Data analysis using different bioinformatics tools

Sample Input and FragmentationThe Genome Sequencer FLX System supports the sequencing of samples from a wide variety of starting materials including genomic DNA,

PCR products, BACs, and cDNA. Samples such as genomic DNA and BACs are fractionated into small, 300- to 800-basepair fragments. For smaller samples, such as small non-coding RNA or PCR amplicons, fragmentation is not required. Instead, short PCR products amplified using Genome Sequencer fusion primers can be used for immobilization onto DNA capture beads as shown below under "One Fragment = One Bead".

Library PreparationUsing a series of standard molecular biology techniques, short adaptors (A and B) - specific for both the 3' and 5' ends - are added to each

fragment. The adaptors are used for purification, amplification, and sequencing steps. Single-stranded fragments with A and B adaptors compose the sample library used for subsequent workflow steps.

One Fragment = One BeadThe single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads. Each bead carries a unique single-stranded

DNA library fragment. The bead-bound library is emulsified with amplification reagents in a water-in-oil mixture resulting in microreactors containing just one bead with one unique sample-library fragment.

emPCR (Emulsion PCR) AmplificationEach unique sample library fragment is amplified within its own microreactor, excluding competing or contaminating sequences.

Amplification of the entire fragment collection is done in parallel; for each fragment, this results in a copy number of several million per bead. Subsequently, the emulsion PCR is broken while the amplified fragments remain bound to their specific beads.

One Bead = One ReadThe clonally amplified fragments are enriched and loaded onto a PicoTiterPlate device for sequencing. The diameter of the PicoTiterPlate

wells allows for only one bead per well. After addition of sequencing enzymes, the fluidics subsystem of the Genome Sequencer FLX Instrument flows individual nucleotides in a fixed order across the hundreds of thousands of wells containing one bead each. Addition of one (or more) nucleotide(s) complementary to the template strand results in a chemiluminescent signal recorded by the CCD camera of the Genome Sequencer FLX Instrument. For a detailed explanation of this reaction see Sequencing Chemistry.

Data AnalysisThe combination of signal intensity and positional information generated across the PicoTiterPlate device allows the software to

determine the sequence of more than 1,000,000 individual reads per 10-hour instrument run simultaneously. For sequencing-data analysis, three different bioinformatics tools are available supporting the following applications: de novo assembly up to 400 megabases; resequencing genomes of any size; and amplicon variant detection by comparison with a known reference sequence.

See http://www.454.com/products-solutions/how-it-works/index.asp

PCR Animation- MCB Chapter 5

• http://bcs.whfreeman.com/lodish7e/#800911__812049__

The Polymerase Chain Reaction (PCR)

•PCR is a cloning method without a host

•Thermus aquaticus, a hot spring bacterium, produces Taq polymerase

•Taq polymerase, unlike E. coli DNA polymerase, is not denatured at 95°C

Chemical synthesis of DNA(oligonucleotide synthesis

using phosphoramidite chemistry)