Diagnostica Molecolare - slide

ESTRAZIONE DNA MEDIANTE KIT DIAGNOSTICO

Overview, continued

Flow Chart

The figure below describes the experimental process.

• Add Solution A and Vortex
• 65°C for 10 minutes
• Add Solution B and Vortex
• Add Chloroform and Vortex
• Centrifuge
• Transfer aqueous phase to new tube
• Add ethanol and precipitate DNA

Materials Supplied by User

The following materials need to be made or supplied by the user. Some reagents apply only to specific protocols, so review the protocol in question before you decide which reagents to prepare.

Reagents

• chloroform
• 100% ethanol
• 80% ethanol
• YPD medium
• SCED solution
• 20% polyethylene glycol in 1 M NaCl
• Phosphate Buffered Saline (PBS; Catalog no. 10010-023)
• Zymolyase
• 1 M Sorbitol, 100 mM EDTA, 14 mM β-mercaptoethanol
• Isopropanol

Equipment

• sterile 1.5 ml microcentrifuge tubes
• shaking water bath
• water bath or heat block
• centrifuge, low-speed
• microcentrifuge
• sterile 15 ml snap-cap polypropylene tubes (Falcon 2059 or equivalent)
• vortex
• ice bucket with ice

Expected Yields

The following table gives the approximate yield of DNA from a variety of samples.

*Not determined, but the DNA yields a discrete PCR product using primers specific for the polyhedrin gene.

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Overview

Easy-DNA^TM Kit

The Easy-DNA^TM Kit is a simple, quick, and inexpensive method for the isolation of DNA from a variety of sources. DNA from the sources listed below has been successfully isolated and used to produce PCR products or for Southern blot experiments.

• fresh, dried, frozen, or heparinized blood
• tissue culture cells, both suspended and trypsinized
• mammalian tissue
• E. coli
• yeast cells
• plant leaves
• hair follicles
• mouse tails
• baculovirus (viral particles)

RNA

Step 1: extract FCoV RNA (containing FCoV genetic code)

Step 2: make DNA copy of genes using the enzyme reverse transcriptase

Step 3: make millions of copies of the FCoV genes using an enzyme called Taq polymerase

Step 4: visualise on gel: positive samples give bright bands

Pos. Pos. Pos. Markers

Step 2: make DNA copy of genes using the enzyme reverse transcriptase

Step 3: make millions of copies of the FCoV genes using an enzyme called Taq polymerase

Step 4: visualise on gel: positive samples give bright bands

Pos. Pos. Pos. Markers

POLYMERASE CHAIN REACTION - PCR

Sequence to amplify

Heat to separate Cool, add primers

Add Taq polymerase

Heat to separate Cool, add primers

Heat to separate Cool, add primers

Add Taq polymerase

From these, get amplifications of the specific target sequence

Repeat cycles

Polymerase Chain Reaction (PCR)

• Template
• Melt DNA
• Anneal Primers
• Extension
• Products

Apparecchi PCR

Analisi elettroforetica dei prodotti PCR

Analisi elettroforetica di prodotti PCR

Analisi degli eteroduplex

Wild type

Mutant

Heteroduplexes

Homoduplexes

mV

Retention time (min)

57 °C

DHPLC per l’analisi degli eteroduplex

DHPLC per l'analisi degli eteroduplex

WAVE Low-Range Mutation Standard

• 52°C
• 53°C
• 54°C
• 55°C
• 56°C
• 57°C
• 58°C
• 59°C

Time (Minutes)

Absorbance (mV)

DHPLC per l’analisi degli eteroduplex

0701/1412

02-2-1-8

0701/1405

02-2-1-9

02-2-1-8/9

exon 3

DHPLC per l’analisi degli eteroduplex

homozygous samples: one peak

heterozygous samples: characteristic peak profiles at selected temperatures

ideally 4 separated peaks, observed profiles strongly depend on temperature and DNA sequence.

For a review see: Xiao and Oefner Hum. Mutat. 17 (2001) 439.

Primer for replication

Strand to be sequenced

Primed DNA

Prepare four reaction mixtures; include in each a different replication-stopping nucleotide

• C
• G
• A
• T

Replication products of "C" reaction

Separate products by gel electrophoresis

Read sequence as complement of bands containing labeled strands

Figure 1. The Sanger sequencing reaction.

Single stranded DNA is amplified in the presence of fluorescently labelled ddNTPs that serve to terminate the reaction and label all the fragments of DNA produced. The fragments of DNA are then separated via polyacrylamide gel electrophoresis and the sequence read using a laser beam and computer.

Figure 2. An electropherogram of a finished sequencing reaction. As the fragments from the sequencing reaction are resolved via electrophoresis, a laser reads the fluorescence of each fragment (blue, green, red or yellow) and compiles the data into an image. Each colour, or fluorescence intensity, represents a different nucleotide (e.g. blue for C) and reveals where that nucleotide is in the sequence.