Nested PCR,Fusion primers, Integrated PCR

Fusion primer and nested integrated PCR( FPNI-PCR): a new high-efficiency strategy for

rapid chromosome walking or flankingsequence cloningZhen Wang, Shafei Ye, Jingjing Li, Bo Zheng, Manzhu Bao and Guogui

NingBioMed Central(BMC) BiotechnologyVolume No. 11 :1092011

Presented by:

Fouzia Bibi-003 Beenish Majeed-009

Ayesha Liaqat-005

Beenish Majeed-009

Genomic DNA

extraction

Oligonucleotide Primers

PCR cond. &

Procedure

Gel Analysis

DNA Sequencing

METHOD

Extraction of Genomic DNA of seven “Rosaceace” species.

Genomic DNA of tobacco, Petunia hybrida, Arabidopsis and rice. [20]

From young leaves by NaOH fast extraction. [21]

Genomic DNA Isolation

Sequence specific primers designed by primer5 software. Annealing temperature (Tm) 60˚C to 72˚C. Arbitrary degenerate (AD) primer designed. AD oligos were fused to 3’ end of adaptor oligo form Fusion

primers (FPs).

Type I primers (simplest form)Type II primers (hairpin structure at 5’ end of single stranded

adaptor regions)Type III (contain 4-6 fixed nucleotides at 3’end)

Annealing temp (Tm) 36˚C to 48˚C.

Oligonucleotide Primers

First Stage

2.0 µL DNA extraction soln. 0.2 µL SP1, 1.0 µL FAD primers and 1 U rTaq, total vol. 20 µL.

Annealing temp. between 28˚C and 52˚C in 9-18 cycles.

Second Stage

1.0 µL 1st step product, SP2 and FSP1 primers, total vol.20µL

1-fold diluted PCR products

30 High stringency cycles employed

Third Stage

1.0 µL 2nd step products, SP3 and FSP2 primers, total vol.20 µL

50-fold diluted PCR products

12 High stringency cycles

PCR Conditions and Procedure

Gel analysis DNA sequencing 5-8 µL separated by

electrophoresis in 1.5% agarose TAE gel

Stained with ethidium bromide

Observed under UV illumination system

• 12-15 µL products purified using Sephadex G-50 column

• Sequencing using SP3 primers designed to known sequence

Direct Sequenci

ng

• Bands of largest products excised from agarose gel

• Purified, cloned into pMD18-TA cloning vector

• Sequenced with M13 forward or reverse primers

Indirect Sequenci

ng

Results and Discussion

Using FPNI-PCR, isolated 21 genomic sequences .

Nine fusion primers in conjunction of sequence-specific primers.

These gene sequences deposited into Genbank library.

Amplify various lengths of products expected flanking regions of 3 different gene of rice and Arabidopsis.

Small DNA molecular size of vector flanking DNA successfully amplified.

Amplified DNA fragments showed 100% accuracy to target products.

Effectiveness and Accuracy

Amplified Products of 3rd Round

(a). Cloning of the FT-Like gene of Rosa rugosa using 3 gene-specific and 1-8 FP primers. (b).T-DNA flanking sequence cloning 3 transgenic tobacco individual plants using forward primers ,various fragment sizes were obtained (c).T-DNA flanking sequence cloning using backward primers. Only target specific fragments were amplified

Ayesha Liaqat-005

Comparative AnalysisTAIL-PCR FPNI-PCR

Products of TAIL-PCR ,FT ortholog cloning of pyracantha fortuneana obtained 510 bp fragment using 4th AD primer only

Products of FPNI-PCR , FT ortholog cloning of pyracantha fortueana four specific fragments amplified, obtained 1.7kbp

Cont…..TAIL-PCR FPNI-PCR

Products of TAIL-PCR, in SOCI ortholog cloning of pyracantha fortuneana, there is situation of “off target”

Products of FPNI-PCR, SOCI ortholog cloning of pyracantha fortuneana , obtained two fragments of 2068 bp and 683bp.

Rapid identification of target genomic sequence, reducing input of time and effort

No difference found between direct sequencing and indirect sequencing of cloned products within vector

Can be proceed 1st step to 2nd without prior dilution of products

In 2nd PCR reaction can achieve > 85% positive cloning of target products

Efficiency of FPNI-PCR

Amplify target products using 42-45 cycles, completed less than 3 hours from start of extraction

Proceeding through 3rd PCR & final sequencing steps, provide 100% positive results

Total procedure was completed in just 54-57 cycles, in less than 4 hours

Cont……

FPNI-PCR strategy of Amplification

3’ section of the FP primers (FPs) maximize thelevel of similarity with common genomic oligosequences

Universality of the designed fusionprimers

Only target DNA amplified via high stringency cycles, Non target products are not amplified by the gene-specificand FP-specific primer pairs

Significant differences

TAIL PCR Yield small DNA fragments

2 rounds of laborious DNA dilutions

Uneconomical for primer designing

Just 60% of reactions yielded specific products [3]

Take a whole working day

Demand only extremely modestlevels of quantity and purity of the template DNA

FPNI PCR FPNI-PCR reactions yielded DNA

fragments larger than 1.0 kb [6]

Less complicated and easy to manipulate

100% specific product amplification through high and low annealing T [1,3]

42-45 cycles take only 2 hours

Demand only extremely modestlevels of quantity and purity of the template DNA

Cont…Inverse and ligation-mediated PCR-based genome walking methods relies on the restriction cleavage, ligation, or tailing before PCR amplification

The procedures of integrated restriction cleavage, ligation and tailing make the Genome Walker Kit a substantially more complicated and time-consuming process than the first PCR step of FPNI-PCR. [15,18]

1. [1] Liu YG, Mitsukawa N, Oosumi T, Whittier RF: Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. The Plant Journal 1995, 8:457-463.

2. [2] Ji JB, Braam J: Restriction Site Extension PCR: A Novel Method for HighThroughput Characterization of Tagged DNA Fragments and Genome Walking. Plos One 2010, 5:1-5.

3. [3] Liu YG, Chen YL: High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. BioTechniques 2007, 43:649-656.

4. [4] Reddy PS, Mahanty S, Kaul T, Nair S, Sopory SK, Reddy MK: A highthroughput genome-walking method and its use for cloning unknown flanking sequences. Analytical Biochemistry 2008, 381:248-253.

5. [15] Liu FY, Ma B, Zhao Y: Characterization of the gene encoding glycoprotein C of duck enteritis virus. Virus Genes 2008, 37:328-332.

6. [20] Yang C, Zhang J, Xu Q, Xiong C, Bao M: Establishment of AFLP technique and assessment of primer combinations for Mei Flower. Plant Molecular Biology Reporter 2005, 23:790-791.

7. [21] He YH, Ning GG, Sun YL, Qi YC, Bao MZ: Identification of a SCAR marker linked to a recessive male sterile gene (Tems) and its application in breeding of marigold (Tagetes erecta L.). Plant breeding 2009, 128:92-96

References

1. [20] Yang C, Zhang J, Xu Q, Xiong C, Bao M: Establishment of AFLP technique and assessment of primer combinations for Mei Flower. Plant Molecular Biology Reporter 2005, 23:790-791.

2. [21] He YH, Ning GG, Sun YL, Qi YC, Bao MZ: Identification of a SCAR marker linked to a recessive male sterile gene (Tems) and its application in breeding of marigold (Tagetes erecta L.). Plant breeding 2009, 128:92-96

Cont…