Oscar Andrés Parra PinoJuan Sebastián Parada Zuluaga

Medicine students

Molecular Biology

INTRODUCTION

Obtain a set of genetic elements identical to its

precursor

Acellular cloning =

Amplification in vitro of DNA or RNA molecules

Protein chain reactionPCR

Without cell intervention

INTRODUCTIONDevelopment of an efficie

nt DNA

assembling

strategy

• Using ligation products as template benefited by full length PCR

adventages

• rapid and easy performance of gene splicing• Less time and less effort to obtain the assembled full

lenght DNA

Class lls

endonuclease

• used in DNA splicing by direct ligation and the assemble DNA fragments through well-designed primers by PCR.

PCR Polymesare chain reaction

PCR: Kary Mullis (1980)Diverse mixtures of different sources

DNA fragments, genes, RNA

Amplification! Enzymatic process that increase number of copies of particular DNA sequence

Low concentration DNA fragments

Milions of copies

Short time

DNA fragments Alignment

hibridation

Primers and dNTPAt 37 – 63°c

DNA denaturation

SynthesisReplication by DNA

polymeras 5´ - 3´At 72 – 75°c

Heating reaction 90- 97°c

PCR Polymesare chain reaction

What´s necessary?

DNA with it region to be amplified Primer: hibridaze with known specific region of DNA dNTPs: ATP, GTP, TTP, CTP Buffer solution MgCl2

DNA polymerase •Thermostable•Usually TAC polymerase: thermus aquaticus

PCR Polymesare chain reaction

Characteristics of the process

• Performance

• Fidelity

• Duration

• Detection capability

• Specificity

PCR Polymesare chain reaction

UTILITY

PCR applications are numerous and diverse.

Cloning of DNA fragments

Evolution studies

Sequencing of nucleic acids

Mutation tracking

DNA characterization for transplantation

PCR Polymesare chain reaction

UTILITY

Prenatal diagnosis of genetic diseases

Forensic medicine

Establishment of polymorphism

Detection of tumor cells and infectious microorganism

Sequence detection with no preliminary purification

PCR Polymesare chain reaction

OBJECTIVE

Development of an efficient strategy

to perform a rapid and easy gene

splicing, especially in various process

when more fragments need to be

assembled at the same time.

1. Extracción de ADN genómico:Conidio de Aspergillus niger CGMCC 3.4628 •Inuculada en Agar papa dextrosa (APD) y posteriormente incubada, según los protocolos.

Utilidad:•Presencia de mutaciones•Clonaciones •Evaluar el manejo de ciertas patologías, generalmente por infecciones.

MATERIALES Y MÉTODOS

2. Diseño del primer:

INTERNOS• 2-3 bases de nucleotidos

protectores• Sitio de reconocimiento de ECIIs• Sitio de esicion de EcllsGen de la region específica

EXTERNOs• primer F1• primer R6

MATERIALES Y MÉTODOS

Disminuyendo la temperatura, servirán de

molde para que DNA polimerasa TAC elongue la cadena hacia el extremo 3´ usando nucleótidos libres, a

una T° = 72°C

MATERIALES Y MÉTODOS

3. PCR segmentada

Este proceso se lleva a cabo

usando el DNA genómico

como molde y con diversos

primers/cebadores.

MATERIALES Y MÉTODOS

4. Digestión enzimática

Las endonucleasas digirieron los amplicones:

FastDigest Ecoll, EcolHindlll

MATERIALES Y MÉTODOS

5. Ligamiento por T4 DNA ligasa

Los productos de ADN digeridos fueron mezclados juntos y posteriormente ligados con LigaFastTM (sistema rápido de ligamiento de DNA)

MATERIALES Y MÉTODOS

6. PCR directa

Para lograr la longitud total ensamblada de ADN, el proceso se realiza con

2ul de productos de ligación como molde y cebadores externos (F1 y R6)

en 50ul de solución estándar de PCR que contiene 1.254u de Primestar SA

ADN polimerasa.

Con este tipo de PCR se superan los límites que presenta la PCR

convencional, para lograr la amplificación del material con completa

fidelidad para un determinado objetivo.

RESULTADOS

El proceso general del proceso experimental se puede resumir en

cuatro pasos

• PCR segmentada para obtener Exones separados

• Digestión de los fragmentos con enzimas de restricción

• Unión de los fragmentos en una sola cadena formando el gen

deseado

• PCR de la secuencia completa obtenida del gen para

amplificarla

RESULTADOS

• Finalizando el proceso, se realizó una electroforesis en gel de

agarosa para cada uno de los exones replicados y para los

fragmentos amplificados del gen completo luego de

ensamblado, este último mostró tener el tamaño en pares de

bases anticipado, confirmando así la unión exitosa de los

fragmentos.

RESULTADOS

DISCUSION Author Method employed New methodLebedenko et al., 1991 Segmented PCR products were cloned into

intermediate cloning vectors.Segmented PCR products were digested directly through restriction enzymes

The assembled DNA was cloned into pRSET expresion vector, obtaining positive colonies

Assembled DNA was attempted to clone into expresion vectors but failed at obtaining positive colonies

Ligation products required to be separated by electrophoresis and the desiredpolynucleotides were isolated

full-length PCRwas employed directly using ligation products as templates toamplify the assembled full-length DNA fragments

Yan et al., 2012 Segmented PCR products were combined through a single restriction-ligation step

Segmented PCR products were combined through a primary and secondary digestion-ligation reaction

CONCLUSIONSUsing direct full length PCR insead of using cloning vectors its quite a lot faster, but as a disadvantage, the resulting DNA fragmentes can not be stored for a long time

The article mentioned that they tried to clone the assembled DNA intopRSET expression vector but this step failed the cause of this is yet to be found as no explanation was given to this

The new method is specialy useful in the assemble of multiple DNA fragments as previous methods has the problem of sample contamination by undesired ligation

bioproducts

Using primary and secondary steps in segmented PCR, Enzyme digestion and segment ligation was key to the success of the experiment as it what made it more specific in

the formation of the full length DNA fragments

Oscar Andrés Parra Pino

Juan Sebastián Parada Zuluaga

BIBLIOGRAFÍA

• MARTÍNEZ SÁNCHEZ, Lina María. Biología molecular. 7 edición. Medellín:

Universidad Pontificia Bolivariana. Facultad de Medicina. 2012. p: 128.

• Lu-Bin Zhou , Qing-Qing Lin, Jing-Xian Zhang, Shu-Juan Zhao, Zhi-Bi Hu. A

rapid DNA assembling strategy mediated by direct full-length polymerase

chain reaction. Gene 523 (2013) 122–125.

GRACIAS