4.17
Sequenciamento Maxam-Gilbert
No mesmo ano da descoberta do método de sequenciamento Sanger, outro grupo de cientistas, Allan Maxam e Walter Gilbert, demonstraram seu método de cli…
No método Maxam-Gilbert, ou clivagem química, o DNA molde é primeiro desnaturado e radiomarcado na extremidade 5 'com um fósforo 32.
A reação de sequenciamento é executada em quatro tubos de reação diferentes com quatro produtos químicos diferentes com a capacidade de modificar diferentes nucleotídeos no DNA.
Por exemplo, o ácido fórmico atacará apenas as purinas, adenina ou guanina, enquanto a hidrazina atacará apenas as pirimidinas - citosina ou timina. Da mesma forma, produtos químicos como o dimetil sulfato atacam apenas um único nucleotídeo.
A piperidina é então usada para clivar o DNA nas bases modificadas, gerando fragmentos de DNA radiomarcados de diferentes comprimentos.
As quatro reações são então executadas paralelamente em um gel e visualizadas por autorradiografia. A sequência do DNA molde pode então ser decifrada lendo a autorradiografia de baixo para cima
View the full transcript and gain access to JoVE Core videos
Q1: What chemicals are used in Maxam-Gilbert sequencing to target specific nucleotides?
Maxam-Gilbert sequencing uses different chemicals to modify specific nucleotides. Formic acid attacks purines (adenine and guanine), while hydrazine targets pyrimidines (cytosine and thymine). Dimethyl sulfate modifies individual nucleotides selectively. Piperidine then cleaves the DNA at these modified bases, generating radiolabeled fragments of varying lengths for sequence analysis.
Q2: How does the Maxam-Gilbert method separate DNA fragments after chemical cleavage?
After chemical cleavage, the four reaction tubes containing modified DNA fragments are run in parallel on a gel using electrophoresis for separation of DNA fragments. The resulting bands are then visualized by autoradiography, which detects the radioactive labels. The DNA sequence is deciphered by reading the autoradiograph from bottom to top, with each band representing a fragment ending at a specific nucleotide.
Q3: Why is radiolabeling with Phosphorus 32 important in Maxam-Gilbert sequencing?
The template DNA is radiolabeled at the 5' end with Phosphorus 32 to enable detection of the resulting DNA fragments. This radioactive label allows autoradiography to visualize the bands on the gel, making it possible to read the sequence. Without radiolabeling, the small DNA fragments generated during chemical cleavage would not be visible after gel separation.
Q4: What are the main limitations of the Maxam-Gilbert sequencing method?
The Maxam-Gilbert method can resolve only up to 400 base pairs in a single run, which is lower than other traditional sequencing methods like Sanger sequencing. It also requires relatively large amounts of template DNA and uses radioisotopes and hazardous chemicals, making it less favorable for routine laboratory use compared to modern alternatives.
Q5: How does the accuracy of Maxam-Gilbert sequencing compare to Sanger sequencing?
The Maxam-Gilbert method is more accurate than Sanger sequencing because it uses direct purified DNA without enzymatic amplification steps. This direct approach minimizes errors that can occur during DNA synthesis. However, despite superior accuracy, the method's requirement for hazardous chemicals and radioisotopes limits its widespread adoption in modern molecular biology laboratories.
Q6: What are current applications of the Maxam-Gilbert sequencing method?
Although less widely used than modern sequencing techniques, the Maxam-Gilbert method remains preferred for specialized applications including DNA fingerprinting and DNA structural studies. Its accuracy and direct analysis of purified DNA make it valuable for these specific research contexts where precise nucleotide-level information is critical for forensic or structural analysis.
Q7: Why must the template DNA be denatured at the start of Maxam-Gilbert sequencing?
Denaturing the template DNA separates the double helix into single strands, making the nucleotides accessible to the chemical modifying agents. This single-stranded form allows formic acid, hydrazine, dimethyl sulfate, and other chemicals to effectively attack and modify their target nucleotides. Without denaturation, the bases would remain protected within the double helix structure and unavailable for chemical modification.