Differential Display-Reverse Transcription-PCR (DD-RT-PCR) and RNA Ligase mediated-RACE (RLM-RACE)

ByP.R. Rahul, Prasath D, Viswanathan R.

Plant Pathology LaboratoryDivision of Crop Protection

SUGARCANE BREEDING INSTITUTE(INDIAN COUNCIL OF AGRICULTURAL RESEARCH)

COIMBATORE 641007TAMIL NADU, INDIA

Plant Pathology LaboratoryDivision of Crop Protection

SUGARCANE BREEDING INSTITUTE(INDIAN COUNCIL OF AGRICULTURAL RESEARCH)

COIMBATORE 641007TAMIL NADU, INDIA

Crop Improvement DivisionIndian Institute of Spices Research,

Marikunnu P.O., Calicut

Crop Improvement DivisionIndian Institute of Spices Research,

Marikunnu P.O., Calicut

An Overview of gene cloning strategies

No previous information available about gene/s of interest

Differential library screening

mRNA Differential DisplaycDNA-AFLPSerial Analysis of Gene Expression (SAGE)Expressed Sequence Tag (EST) AnalysisMicroarray Analysis

Subtractive Hybridization

Two-Dimensional Gel Electrophoresis (2DE)

Has the gene been cloned from a related species ? / Partial amino acid sequence available

PCR / RT-PCR with Degenerate Primers (RGA scanning)

Screen Genomic / cDNA Library with heterologous probe

Is there a mutant in which the gene is deficient ?

T- DNA TaggingTransposon TaggingTILLING

Any closely linked marker identified ? Map based cloning using BAC or YAC clones

Differential Display-RT-PCR

Differential display of eukaryotic messenger RNA by means of the

polymerase chain reaction.

Liang P, Pardee AB. Science 1992; 257:967-71. (PMID 1354393)

Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions.

This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction.

The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer.

The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel.

When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.

Methodology work flow

DD-RT-PCR

Background of work• Sugarcane- Tropical crop ,

• Family - Poaceae , Genus – Saccharum

• Hybrids - Saccharum officinarum L. (Noble canes), S. sinense (Chinese clones), S. barberi (North Indian clones), and S. spontaneum (wild cane)

• Red rot is one of the oldest most devastating disease of sugarcane

• Red rot -Java, Indonesia - Went (1893) - causal organism as C. Falcatum

• Total loss - red rot epiphytotics (1990’s) in Tamil Nadu

• Barber (1901) and Butler (1914-1918)

• Loss in yield - loss of millable canes, juice quality-impaired sucrose metabolism

• Ratoon crops loss- heavy

• Loss of commercial varieties :Co 285, Co 312, Co 419, Co 449, Co 527, Co 658, Co 997, Co 1148, Co 6304, Co 7805, Co 86002, CoC 671, CoC 85061, CoC 86062, CoC 92061, CoJ 64, CoS 8436 etc. in the country

• Disease control - disease-resistant varieties through breeding - fungicide applications-good cultural practices

• impervious -rind, fibrous -cane nodal region.

• Non availability of water soluble systemic fungicides against Cf, -penetrate setts - kill the pathogen selectively-annulled by rich nutrient present in the sett

Differential Display Analysis

Genhunter

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

Differential Display Analysis

Genhunter

mRNA

DNA- free polyA+ RNA

------AAAA -----------AAAA --------AAAA

Reverse transcribe with anchored oligo dT Primers;cDNA pools result

-------AAAAA ------------AAAAA ------------AAAAA

---GMTTTTTT ------CMTTTTT -----AMTTTTTM=G,C,A(12 combinations of the last two 3’ bases excluding T at the penultimate end,so each oligo dT primer recognises 1/12 of the mRNA population)

Perform PCR:amplify with arbitrary decamers or 20 mers and original anchored oligo dT

NNNNN------------------ NNNNN-------------- NNNNN---------------

----------------GMTTTT ------------AMTTTT -------------CMTTTT

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

Reverse transcribe

Differential Display Analysis

Genhunter

mRNA

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

Anchor Primer (3)

Reverse transcribe

Differential Display Analysis

Genhunter

mRNA

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

Differential Display Analysis

Genhunter

Anchor Primer (3)

Reverse transcribe

mRNA

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

cDNA(s)

Differential Display Analysis

Genhunter

Anchor Primer (3)

Reverse transcribe

mRNA

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

PCR Amplify

cDNA(s)

labeled dNTPs

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

PCR Amplify

Anchor Primer (3)

cDNA(s)

labeled dNTPs

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

PCR Amplify

Arbitrary Primers(80)

cDNA(s)

Anchor Primer (3)

labeled dNTPs

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

PCR Amplify labeled dNTPs

cDNA(s)

Arbitrary Primers(80)

Anchor Primer (3)

X Y Z

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

PCR Amplify

Run on denaturing Polyacrylamide gel

cDNA(s)

Arbitrary Primers(80)

Anchor Primer (3)

labeled dNTPs

X Y Z

Coding region5’ UTR 3’ UTR

UCG

AAAAAAAn

PCR Amplify

cDNA(s)

Arbitrary Primers(80)

Anchor Primer (3)

labeled dNTPs

Run on denaturing Polyacrylamide gel

X Y Z

Autoradiography-X-Ray film/Phosphor imager

Phosphorus-33: It is less energetic than P-32, giving a better resolution. 5118 Ci/mmole.

Phosphorus-32: Its high energy and low half life result in lower autoradiography exposure times. Unfortunatelly its higher energy requires acrylic glass protection and a film badge. 9131 Ci/mmole.

We use P33 labelled in ATPs, after the PAGE gel the GEL is transferred to the 3M whatmann paper and dried in a gel drier or in an oven for 1-2 hours

X-Ray based autoradiography

The Dried gel is then placed over the Kodak Biomax MR film with emulsion side of the film facing the gel

It is placed tightly in a cassette and kept at -80 for 3-5 days exposure

On the fifth day the x-ray is removed from the cassette and developed in dark room using developer 30-60sec, distill water wash 30-60 sec, fixer 30-60 sec and water wash 1 minute

The X-ray is hung on string to dry.

Phosphor imager based autoradiography

• The advantages compared to film are that the exposure times are considerably shorter(usually 5 - 10 times) • Images are in practice very rarely overexposed • The sample is placed in close contact with the imaging screen and exposed analoguously to normal autoradiography

Principle

A phosphor imager is based on imaging plates that replace normal X-ray film in autoradiographic measurements.

An imaging plate consists of a thin layer of special crystals doped with lanthanides. Radioactive radiation excites the crystals and a latent image of the sample is formed on the plate.

The image is read into the computer by scanning the plate with a red laser beam using a dual optical fiber.

The red light further excites the preexcited phosphor particles causing emission of blue light. The intensity of this emission is measured with a photomultiplier and used to construct an image of the sample

The (PSL) photo-stimulated luminescence released upon the laser is collected into the photomultiplier tube (PMT) through the light collection guide and is converted to electric signals. Subsequently, these are converted to digital signals of 8 to 16 bits, again depending on the intended purpose

The Imaging Plate is reusable after erasing the residual latent image with uniformly irradiated visible light

Flexible image sensor in which bunches of very small crystals (grain size: about 5 µm)of photo-stimulable phosphor of barium fluorobromide containing a trace amount of bivalent europium as a luminescence center,formulated as BaFBr: Eu2+, are uniformlycoated on a polyester support film.  

Induced

Induced

Constant

Induced

Induced

Constant

Gel extract

PCR amplify

Gel extract

PCR amplify

Ligate in vector, transform…miniprep and sequence

Gel extract

PCR amplify

Ligate in vector, transform…miniprep and sequence

Spot sequences on arrays

Run products on a sequencing gel

Isolate differentially expressed products (reamplify)

Confirm differential expressionNorthern analysisRT-PCRReverse northern analysis

Clone

Sequence

•SensitivityPCR-based technique, minimal RNA requiredLow abundance target theoretically identified•Many samples may be analysed simultaneouslyQualitative & quantitative

-Technology is fairly simple.-You increase your probability that the clones that you spot on the macroarrays will be differentially regulated.

Advantages

Differential Display AnalysisGenhunter

Disadvantages

- It would be fairly expensive to get hundreds of differentially expressed clones.- Hard to identify the sequences that you clone in the data base using genehunter technology.

• Only fraction of RNA amplified Primers must bind within 500bp of 3’ end Bias towards some transcripts•>50 % false positives

Autoradiography image of differential

display gel of sugarcane cell

suspension upon elicitor treatment

DIFFERENTIAL DISPLAY

~150 transcripts have been cloned and sequenced from field study

Run products on a sequencing gel

Isolate differentially expressed products (reamplify)

Confirm differential expressionReverse northern analysis

Clone

Sequence

Overall flow of DD-RT-PCR

work

DDRT-PCR

Full length isolation of potential transcripts by RACE

Total differential transcripts

Up-regulated transcripts

Down-regulated transcripts

Relative abundance of DD transcripts categorized based on putative functions

RACE - Rapid Amplification of cDNA Ends

The RACE RT-PCR strategy was first described in 1988 by

Michael Frohman and Gail Martin

isolated cDNA clones of mRNA from int-2, a mouse gene (2.9 kilobases)

5’ RACE is as not easy as it sounds.

It needs three sequential enzymatic steps

1. Reverse Transcription (Reverse transcriptase)

2. Addition of homopolymeric tails (Terminal transferase)

3. PCR (DNA polymerase)

Traditional 5' RACE is sometimes successful, but the major limitation of

the procedure is that there is no selection for amplification of fragments

corresponding to the actual 5' ends of mRNA: all cDNAs are acceptable

templates in the reaction. (Truncated cDNA generated during 5’ RACE

have the same termini as full length molecules (poly A in 3’ and

homopolymeric tail at 5’))

• Additionally, the PCR step selects the most efficient amplicons (e.g., the smallest), favoring amplification of less than full-length products. 5' RACE usually produces a heterogeneous population of amplified products.

• Amplified DNA s are of such variable length that they usually appear as smear on an agarose gel

• Specificity of adaptors is too low –some times binding of adaptor primers occur randomly

• RT has difficulty in transcribing 5’ of mRNA due to its high degree of secondary structure in 5’ regions and low fidelity and inactivation of enzymes at higher temperatures.

RNA ligase-mediated RACE (RLM-RACE) developed by Ambion and now available from Ambion, Invitrogen, Roche companies.

• RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) represents a major improvement to the classic RACE technique

• RLM-RACE is designed to amplify cDNA only from full-length, capped mRNA, usually producing a single band after PCR.

• RLM-RACE is optimized for efficiency and reliability

• Involves the use of bacteriophage T4 RNA ligase to covalently attach a single strand RNA anchor molecule to the de-capped 5’ end of mRNA. First strand cDNA synthesis can then be performed using a gene-specific primer to produce a pool of cDNAs encoding the anchored primer sequence.

•Calf Intestine Alkaline Phosphatase (CIP)

Total or poly(A) selected RNA is treated with Calf Intestine Alkaline

Phosphatase (CIP) to remove free 5'-phosphates from molecules

such as ribosomal RNA, fragmented mRNA, tRNA, and

contaminating genomic DNA. The cap structure found on intact 5'

ends of mRNA is not affected by CIP.

•Tobacco Acid Pyrophosphatase (TAP)

The RNA is then treated with Tobacco Acid Pyrophosphatase (TAP)

to remove the cap structure from full-length mRNA

5'-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3'

• involves the use of bacteriophage T4 RNA ligase to covalently attach a single strand RNA anchor molecule to the de-capped 5’ end of mRNA.

• A 45 base RNA Adapter oligonucleotide is ligated - using T4 RNA ligase.

• The adapter cannot ligate to dephosphorylated RNA because these molecules lack the 5'-phosphate necessary for ligation.

* Nested primers

Calf Intestine Alkaline Phosphatase (CIP)

The RNA is then treated with Tobacco Acid Pyrophosphatase (TAP) to remove the cap structure from full-length mRNA

Involves the use of T4 RNA ligase to covalently attach a single strand RNA anchor molecule to the de-capped 5’ end of mRNA

RACE (RLM-GeneRacer kit Invitrogen)

• 3' RACE is generally a much easier procedure than 5' RACE. First strand cDNA is synthesized from either total RNA or poly(A)-selected RNA, using the supplied 3' RACE Adapter

• The cDNA is then subjected to PCR using one of the 3' RACE Primers which are complimentary to the anchored adapter, and a user-supplied primer for the gene-of-interest.

• 3' RACE may not require a nested PCR, but a pair of nested primers for the Adapter sequence are provided in case nested PCR is determined to be necessary.

GeneRacer. RNA oligo sequence

5′-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3′44 bases

GeneRacer. Oligo dT primersequence

5′-GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T)18-3′54 bases

Primer positions

General PCR Primer Design Suggestions

Use the following PCR primer design recommendations:

• 50 to 70 % GC content to allow a high annealing temperature (>72 C).

• 23 to 28 nucleotides to improve specificity of binding.

• Low GC content at 3’ends to minimize extension by DNA polymerase at nontarget sites (with no more than two G or C residues in the last five bases).

• No self-complementary sequences in the primer or no sequence complementary to the primers supplied with the GeneRacer- cDNA, especially at the 3’ end.

• An annealing temperature greater than 72 C Using primers with a high annealing temperature improves the specificity of the RACE PCR, and it allows use of touchdown PCR

Touchdown PCR :

•developed by Don et al in 1991

•The initial cycles of touchdown PCR has annealing temperature higher than the Tm of primer pair and decrease gradually until it reaches Tm.

•The annealing temperature is decreased by 1°C every cycle or every second cycle until a specified or 'touchdown' annealing temperature is reached.

• The touchdown temperature is then used for the remaining number of cycles.

• Can be used at high annealing temperatures

• it will increase primer binding specificity

• reduce non-specific amplification in early PCR cycles.

BamH1 sites at their 5' end (CGCGGATCC)

• inner gene specific primers also have restriction sites at their 5' end, PCR fragments generated in the “inner” PCR reactions can be ligated into a digested plasmid vector using standard cloning techniques.

•T/A overhangs to facilitate cloning can be used to clone reaction products without restriction enzyme sites.

RACE amplified products –

lane 1 & 2 represents 14-3-3 like protein

3 & 4 represents flagellar adenylate kinase like protein

M 1 2 3 4

14-3-3 like protein

~1.1 kb fragment

• Mediates signal transduction by binding to phosphoserine- containing proteins.

• They are involved in growth factor signalling and also interact with MEK kinases

Presence of ORF in 14-3-3 sequence

"Hot-start" PCR

•a modification of conventional PCR that reduces non-specific product amplification.

•The hot start PCR method minimizes mispriming and extension.

•In this procedure amplification cannot occur until the reaction temperature is above that where non-specific annealing of primers to targets occurs.

•This block in amplification is usually accomplished by using a DNA polymerase that is inactive until higher temperatures are reached.

In a hot start, a reaction component which is essential for

polymerization, is excluded from the reaction mixture. After heating the

reaction mix above the temperature at which mispriming can occur, the

missing component is added into the reaction mixture, in a small volume.

This method is laborious and prone to cross-contamination.

• Hot start has been developed in which the first part of the reaction mixture is covered with melted wax, which hardens at room temperature and separates the components from the second part of the reaction mixture

• Enzyme preparations contain antibodies to their polymerases to prevent nonspecific amplification.

• The initial denaturing step of the PCR denatures the antibodies, releasing active polymerase into the reaction.

• Withholding a key component of the reaction (e. g., thermostable polymerase or magnesium) until the denaturation temperature is reached.

Applications

• RACE procedures have been used for amplification and cloning of rare mRNAs

• Additionally, RACE may be applied to existing cDNA libraries Random hexamer primed

• cDNA has also been adapted to 5' RACE for amplification and cloning of multiplegenes from a single first strand synthesis reaction

• Products of RACE reactions can be directly sequenced without any intermittent cloning steps or the products can be used for the preparation of probes

• Products generated by the 3' and 5' RACE procedures may be combined to generate full-length cDNAs

• Lastly, the RACE procedures may be utilized in conjunction with exon trapping methods to enable amplification and subsequent characterization of unknown coding sequences.