4.17
Sequenziamento di Maxam-Gilbert
Nello stesso anno della scoperta del metodo di sequenziamento Sanger, un altro gruppo di scienziati, Allan Maxam e Walter Gilbert, dimostrarono il lor…
Nel metodo Maxam-Gilbert, o scissione chimica, il DNA stampo viene prima denaturato e radiomarcato all'estremità 5' con un fosforo 32.
La reazione di sequenziamento viene eseguita in quattro diverse provette di reazione con quattro diverse sostanze chimiche con la capacità di modificare diversi nucleotidi nel DNA.
Ad esempio, l'acido formico attaccherà solo le purine, l'adenina o la guanina, mentre l'idrazina attaccherà solo le pirimidine - citosina o timina. Allo stesso modo, sostanze chimiche come il dimetilsolfato attaccheranno solo un singolo nucleotide.
La piperidina viene quindi utilizzata per scindere il DNA alle basi modificate, generando frammenti di DNA radiomarcati di diverse lunghezze.
Le quattro reazioni vengono quindi eseguite parallelamente su un gel e visualizzate mediante autoradiografia. La sequenza del DNA stampo può quindi essere decifrata leggendo l'autoradiografia dal basso verso l'alto
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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.