Issuu on Google+

Amplification Isolation and purification

Sample collection

Clean up

Detection

Sample handling and preparation within the genomics workflow Chapter 1: Dried biosample collection with Whatman™ FTA™ card technology 1


Sample handling and preparation within the genomics workflow

Index Chapter 1

Dried biosample collection with Whatman™ FTA™ card technology

Chapter 2

Ready-To-Go and amplification techniques

Chapter 3

Fluorescent labelling and detection of biomolecules

2 PowerPoint Style Guide June 2010


Sample handling and preparation within the genomics workflow

Welcome to our ebook, we hope you find it enjoyable and informative. Across three chapters, the ebook introduces some key sample handling and preparation stages within the genomics workflow; particular attention is given to sample collection, ambient temperature stabilisation, amplification and fluorescent labelling and detection. Each chapter will provide a unique insight, with useful practical information and guidelines and is suited to those researchers involved in genomics research and assay development. To take part in the discussion follow us at @xxxxx and visit our blog at www.gelifesciences.com/dxcomponents


Dried biosample collection


Biosample collection

Consideration for biosample collection has to be given to the following (a) collection (b) transportation (c) storage (d) processing

Current practices include (a) specialist collection (b) refrigerated transport (c) refrigerated, freezer or cryogenic storage (d) downstream processing for DNA, protein and or metabolyte analysis

This cold chain logistical workflow, which can be expensive both to establish and maintain, is essential for biosamples where maintaining a sample’s integrity over time is a key requirement.

5 GE Title or job number 6/28/2013


Dried biosample collection Many of the issues associated with the transportation, stabilisation and storage of biological samples can be alleviated by using dried biosample collection.

Dried biosample collection In this technique a biological sample ,

The most common type of biological sample collection is Dried Blood Spot (DBS) collection.

such as blood, serum, buccal cells,

It has been widely employed in neonatal

urine or tissue homogenate is

screening programs for the screening of

transferred to a paper substrate and

metabolic diseases, following the publication in

subsequently dried. The biological

1963 of its application in the detection of

molecules of interest present within

phenylketonuria (PKU) in large populations of

the sample are then extracted for

newborn infants (Guthrie & Susi, 1963).1

subsequent analysis

6 GE Title or job number 6/28/2013 •Guthrie and Susie, Pediatrics 1963; 32:3 338-343


Dried biosample collection

Dry sample collection will aid in the security of supply of biosamples and in key aspects of sampling handling reducing risk

Safer The technique is suited to changing technology and user, patient needs

Future

Dried biosample collection

Easier

Simplified collection & logistics enables collection in and transport from remote & difficult environments as well as more convenient locations

Strips out cost & steps from an existing wet biosample collection & processing workflow

Leaner

7 GE Title or job number 6/28/2013


Whatman FTA card technology Collect, transport, purify and store DNA all at ambient temperature


Dried biosample collection GE Healthcare offers a broad portfolio for the collection and ambient temperature transport and storage of a range of biosamples.

903 collection cards

Whatman FTA cards Chemically coated matrices that provide

Whatman™ FTA™ cards

903™ collection card

Proven for blood collection & widely used in neonatal screening programs

long term stability of DNA at ambient temperature

CE marked for the collection of blood • Lyse cells on sample application

samples from neonates and/or adults by

• Stability: DNA analysis of 22 year-old

healthcare professionals.

blood & 12 year-old buccal samples • FDA-registered in vitro Class II

• Simpler biosample collection

Medical Device

workflow

• Compliant with Directive 98/79/EC

• Eliminate the need for refrigeration and cold chain logistics

Collection for DNA analysis

Collection for protein analysis

• CE marked as an in vitro Diagnostic Medical Device in Europe • 903 Protein Saver Card is a "research use only" format.

9 GE Title or job number 6/28/2013


Whatman™ FTA™ card technology: two different chemistries

Whatman FTA technology Whatman FTA: use when DNA needs to be

Whatman FTA Elute: use when DNA is required in solution

associated with the card/punch

Yields pure DNA in solution, with a simple protocol which

DNA remains tightly bound to the matrix while

eliminates the need for a DNA purification kit

cell membranes and organelles are lysed and proteins and inhibitors are washed away

Whatman FTA

Whatman FTA Elute

• Collect sample and capture DNA in 1 step

• Compatible with downstream applications such as Q-PCR. • Supports small sample volume needs (12-125 μl) • DNA in 15-30 minutes

• Purify DNA in less than 30 minutes • Compatible with a wide range of samples

DNA remains on card

Yields DNA in solution

Associate & Store

Rapid analysis

10 GE Title or job number 6/28/2013 TN


Dried biosample collection: which is the correct card for the collection of DNA?

What is your biological sample?

Coloured

Plant

Clone

Colourless

Is DNA required in solution or associated with card?

FTA Plant Saver

FTA CloneSaver

Is DNA required in solution or associated with card?

Trial our online decision tree to select the In solution

optimal card for your application at

With card

With card

In solution

www.gelifesciences.com/dxcomponents

FTA Elute Micro

Number of sample spots per card?

1

FTA Micro

2

FTA Mini

IND FTA Elute Micro

Number of sample spots per card?

4

1

FTA Classic

FTA Micro

2 FTA Mini

4 FTA Classic


Dried biosample collection: Whatman FTA cards described in >400 peer reviewed publications

# Publications

Sample type

Animal Biotechnology

Blood Blood & other

Biobanking 7%

6%

6%

15%

2%

Dx

6% 4%

Food & beverage 25%

Genomics Environmental

29%

8%

2% 8%

Forensics 3%

Cervical

5%

Buccal 43%

2%

Buccal & blood Culture

3%

Stool

8% 4%

Food & beverage 4%

8%

Human fluids Plant

Pharmacogenomics

Plasmid

Plant biotechnology

2%

Tissue Other*

Has an FTA card been used in your application? By classification 4% 3% 1% 1% 3%

6%

3% 3%

4% 49%

8% 6%

3%

Human

These three charts provide an illustration of how the technology has been used in

Canine

several hundred peer reviewed publications. From diagnostics to plant

Bovine

biotechnology, from human to fungal samples the ease of sample collection with

Ovine

FTA technology and compatibility with downstream analysis techniques has

Porcine

endeared the technology to a range of disciplines.

Animal -other*

4%

Plant 1%

Virus 1%

Search our online database for publications relevant to your application at www.gelifesciences.com/dxcomponents

Fungus Yeast

12 GE Title or job number 6/28/2013

Avian

n = 404 selected publications


Dried biosample collection: how do you prepare DNA from Whatman FTA cards?

Whatman FTA

Whatman FTA Elute

Apply sample to FTA card

Apply sample to FTA Elute card

Three simple steps to pure DNA: spot, punch and Punch from card

purify in less than 30 minutes

40 Âľl of blood produces Wash with FTA Purification reagent

an 8mm spot

This procedure yields pure DNA within 30 minutes of punching from the card, with <5 minutes hands on time.

3mm punch yields >100 ng Wash with aqueous buffer

Punch from card

DNA, suitable for PCR applications

Wash with water and decant

Heme protein is retained on Elute paper and does not appear in solution

Add water & heat to 95oC

Multiple aliquots of eluted DNA can be used in PCR and Add PCR master mix directly to punch

quantitative PCR applications

2 Âľl aliquot for PCR

13 GE Title or job number 6/28/2013


Dried biosample collection: Whatman FTA cards and your whole genome amplification workflow

Wash punch

WGA of gDNA dissociated from FTA by pH denaturation M

1 2 3 4

Denature using KOH

Neutralise using HCl

Discard punch

Add to GenomiPhi reaction

GenomiPhi V3 Control

5 6 7 8 9 10 11 12 13 14 15 16

Genomic DNA was amplified from dried blood stored on FTA classic card after dissociation by pH denaturation (Lanes 1,3,5,7,9,11). Amplified DNA was subsequently subjected to digestion with EcoR1 restriction enzyme (Lanes 2,4,6,8,10,12). Control GenomiPhi V3 amplifications were carried out on purified blood DNA (lanes 13,15) and also subjected to the same restriction digests (lanes 14, 15)

STR analysis of genomic DNA amplified from FTA spotted blood 14 GE Title or job number 6/28/2013


Sample collection with Indicating Whatman FTA Elute cards for a HPV cervical cancer screeen

Current practice in cervical cancer screening programs If +VE

Gynaecologist

Cytologist

HPV test

Gynaecologist

Colposcopy

A national cervical cancer screen programme in a developed economy has the following workflow 1) Woman patient has cervical smear performed by a trained professional. 2) The professionally collected cell sample is analysed by the cytologist. 1) If +ve, patient is referred to gynaecologist 2) If -ve, no further need for gynaecologist 3) If inconclusive or shows an indeterminate results, then a HPV test performed. 4) If HPV test is +ve for any of the high risk types, patient sees gynaecologist & colposcopy

If abnormal

In a future environment, the HPV test may become the primary screen. In this case, a specific professional sample collection is not required, making it possible for the patient to collect the sample herself.

Potential future practice

Using an Indictaing FTA Elute card the sample could be collected remotely and sent for lab analysis in the conventional post. Home collect with FTA Elute card

Post in mail

HPV test

Gynaecologist

If +VE

Colposcopy

Only if the sample is positive would the patient visit the doctors for a secondary screen of cytology and then if necessary a colposcopy The use of FTA cards in this manner could simplify workflow, reduce healthcare costs, increase access and expand patient base 15

Cytologist

GE Title or job number 6/28/2013


Dried biosample collection: simplified DNA purification workflow

Wet sampling

Dried biosample collection

Sample collection with Whatman FTA Elute eliminates the need of a Whatmanâ&#x201E;˘ FTAâ&#x201E;˘ Elute

DNA purification kit, as DNA is eluted in solution following a simple water wash and a heat step.

Time

1 hour

30 mins This reduces the time and number of steps taken to purify DNA from your biological sample

Steps

16 GE Title or job number 6/28/2013


Dried biosample collection: safer sample handling Wet sampling

Dried biosample collection

Consider that a biosample is not viable when it is dried1, unlike when wet and therefore safer than a liquid sample. As such these active biohazards require much stricter conditions of care

Handling

Consider the security of supply when shipping biosamples refrigerated or

Transportation

on dry ice on and the consequences of failure in the cold chain logistics workflow.

Storage

Active biohazards

Disposal

17 GE Title or job number 6/28/2013 1 This is publicly stated by the CDC and the US Postal Service follows this guideline.


Dried biosample collection: transportation cost savings

$32 Studies have shown that 0

10

20

Cost ($)

30

with the transport of a specific blood sample, a 94% cost reduction can be

Wet sampling

experienced in associated dry ice, packaging and documentation

$2

0

94% Saving

10

20

Cost ($)

30

Dried biosample collection

18 GE Title or job number 6/28/2013


Dried biosample collection: some of your questions answered

How many punches can I get from one FTA sample circle? Using the 1 inch circle on the FTA Classic, Mini, Micro Cards, approximately 140 - 1.2 mm discs can be obtained, and over 50 of the 2.0 mm discs. What sample types can I apply to FTA paper? A wide range including: tissue cells, blood cells (human and animal), bacteria, plant cells, liver cells, buccal cells, urine, amniotic fluid, food samples, plant (leaf presses and homogenate), and tumor cells.

What size PCR amplicons can be generated with FTA Elute? Using the standard procedure of heating punches to 95-98째C for 30 min. followed by vortexing, it is possible for up to 1500bp PCR products to be amplified.

What is the typical yield from a 3mm punch of blood samples from FTA Elute? ~30ng, though his will vary on a number of factors including % of nucleated cells in the blood and the hematocrit levels.

What techniques are FTA and FTA Elute compatible with? FTA compatibility includes PCR, Whole genome amplification and sequencing FTA Elute compatibility includes: PCR, QPCR and sequencing Is it safe to mail FTA & FTA Elute cards with sample applied to them? Yes, the cards have been approved for shipping through the U.S. Postal Service. The FTA technology is designed to protect DNA, even in transport. Make sure the card is in a protective envelope to ensure safe transport.

19 GE Title or job number 6/28/2013


Dried biosample collection: questions for you

How are you shipping your biological samples? Did you find the content useful? How are you storing your biological samples?

Would you like to sign up for the remaining chapters in this ebook series? Please click here

How are you purifying DNA from your biological samples?

20 GE Title or job number 6/28/2013


Dried biosample collection: further information Product ordering information

Product literature Product

Code

Product

Code

Whatman™ FTA™ Classic card 100 pk

WB120205

Indicating Whatman FTA Classic card 100 pk

WB120206

Whatman FTA Micro card 100 pk

WB120210

Indicating Whatman FTA Micro card 100 pk

WB120211

Whatman FTA Mini card 100 pk

WB120055

Helping you build a smarter diagnostic assay A comparative performance evaluation of illustra™ Ready-To-Go™ GenomiPhi™ V3 and illustra GenomiPhi V2 DNA amplification kits STR amplification of DNA from blood and buccal samples using FTA technology CloneSaver™ Supports TempliPhi™ Rolling Circle Amplification

Indicating Whatman FTA Mini card 100 pk

WB120056

Indicating Whatman FTA Elute Data File

51716

Indicating Whatman FTA Elute Micro Card 25 pk

WB120412

Whatman FTA Data File

51613

Indicating Whatman FTA Elute Micro Card 100 pk

WB120411

Whatman FTA Elute Micro Card 25 pk

WB120401

Whatman FTA Elute Data File 28-9844-02 AA Instructions for Buccal Cell Collection and Transfer to Indicating FTA™ Cards 29-0055-82 AA

Whatman FTA Elute Micro Card 100 pk

WB120410

Whatman FTA PlantSaver

WB120065

Post-mortem collection and archiving of DNA on Whatman FTA cards

28-9844-37 AA

Whatman FTA CloneSaver™ card

WB120028

Your forensic samples, our experience

29-0055-83 AA

29-0451-02AA

29-0245-46 AA 29-0082-33 AA 28-9843-64 AA

www.gelifesciences.com/whatman www.gelifesciences.com/dxcomponents

21 GE Title or job number 6/28/2013


GE and GE Monogram are trademarks of General Electric Company. Whatman, FTA and 903 are trademarks of GE Healthcare companies. GEHC is the provider of the sample collection device and currently makes no claim for its suitability for a disease specific test The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular Systems and F Hoffmann-La Roche Ltd. All third party trademarks are the property of their respective owners. FTA is a trademark of GE Healthcare companies. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your local GE Healthcare representative for the most current information. Š 2013 General Electric Company â&#x20AC;&#x201C; All rights reserved. First published March 2013 GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK


Fta ebook