A rapid dna assembling strategy mediated by direct full length
1. Oscar Andrés Parra Pino
Juan Sebastián Parada Zuluaga
Medicine students
Molecular Biology
2. Obtain a set of genetic
elements identical to its
precursor
Acellular cloning
=
Amplification in vitro of
DNA or RNA molecules
Protein chain reaction
PCR
3. Development of
an efficient 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.
4. 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
6. 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
7. Characteristics of the process
• Performance
• Fidelity
• Duration
• Detection capability
• Specificity
8. UTILITY
PCR applications are numerous and diverse.
Cloning of DNA fragments
Evolution studies
Sequencing of nucleic acids
Mutation tracking
DNA characterization for transplantation
9. UTILITY
Prenatal diagnosis of genetic diseases
Forensic medicine
Establishment of polymorphism
Detection of tumor cells and infectious microorganism
Sequence detection with no preliminary purification
10. 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.
11. 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.
12. 2. Diseño del primer:
INTERNOS
• 2-3 bases de nucleotidos
protectores
• Sitio de reconocimiento de ECIIs
• Sitio de esicion de Eclls
Gen de la region específica
EXTERNOs
• primer F1
• primer R6
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
13. 3. PCR segmentada
Este proceso se lleva a cabo
usando el DNA genómico
como molde y con diversos
primers/cebadores.
15. 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)
16. 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.
17. 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
18.
19.
20. • 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.
21.
22. Author Method employed New method
Lebedenko 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 desired
polynucleotides were isolated
full-length PCR
was employed directly using
ligation products as
templates to
amplify 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
23. Using 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 into
pRSET 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
