Question

PLEASE ANSWER ALL PARTS FULLY!

How was liquid hybridization used to compare species relatedness? What is the purpose/significance of Driver versus Tracer DNA? What is Subtractive Hybridization, how/why would you use this technique?

Compare Maxam-Gilbert and Sanger DNA sequencing methods. What are the more descriptive names for these techniques? How are the techniques similar? different? What reagents are used in each reaction? How are the results read? Do you get the sequence of the molecule you started with or the complementary molecule? How does fluorescent cycle sequencing differ from the original Sanger Method?

Answer 1

In order to find the genetic similarity between pools of DNA sequences DNA hybridisation is used. It works by finding out the genetic distance between two organisms. The DNA of one organism is labelled, then mixed with the unlabelled DNA to be compared against. The mixture is incubated to allow DNA strands to separate and form hybrid double-stranded DNA. Hybridized sequences with a high degree of similarity will bind firmly, and more energy is required to separate them. They get separated when heated at a higher temperature this process is known as "DNA melting". Now, the double-stranded DNA is bound to a column and the mixture is heated in steps. At each step, the column is washed and the sequences that melt become single-stranded and wash off the column. The temperatures at which labelled DNA comes off reflects the amount of similarity between sequences.

Reactions between the labelled nucleic acid fragments (tracer) and unlabelled complementary strands of different lengths which are present in excess (driver) are used in studies of genomic sequence organization and transcriptional expression.

Subtractive hybridization is a technique used for identifying and characterizing differences between two populations of nucleic acids. This technique detects the differences between DNA of different genomes and also between cell types where deletionsor various types of genomic substitutions have formed. Subtractive hybridization requires two populations of nucleic acids; the tester (or tracer) contains the target nucleic acid (the DNA or RNA differences that one wants to identify), and the driver lacks the target sequences. Only the sequences in common between the tester and the driver hybridize.

There are five basic steps to subtractive hybridization:

• choosing material for isolation of tester and driver nucleic acids;
• production tester and driver;
• hybridisation;
• removal of driver-tester hybrids and excess driver (subtraction);
• isolation of the complete sequence of the remaining target nucleic acid.

Maxam gilbert sequencing: A sequencing method based on chemical modification of DNA and subsequent cleavage of at specific bases.it involves:

• Chemical modification of DNA
• radioactive labelling at 5’ end of DNA
• Purification of DNA to be sequenced
• Chemical treatment generated breaks into DNA
• Run on gel

Sanger DNA sequencing: It is a PCR based method. It is a modified DNA replication method. Growing chains are terminated by dideoxynucleotides.

Maxam gilbert sequencing: Chemical sequencing

Sanger DNA sequencing: Chain terminating sequencing

These two methods of sequencing are similar as both are used to determine the order of nucleotide bases adenine, guanine ,cytosine and thymine in a molecule of DNA.

Reagents : Maxam gilbert sequencing:

Radioactive label – Poly nucleotide kinase
Base modification – Dimethyl Sulfate, Hydrazine
Cleavage of sugar phosphate backbone- Piperidine

Reagents : Sanger DNA sequencing:

Primer
Taq polymerase
Template ss DNA
dNTPs
ddNTPs

Results read by autoradiography and gel electrophoresis in Maxam gilbert sequencing and by gel electrophoresis in Sanger DNA sequencing.

Cycle sequencing is dideoxy mediated sequencing using PCR and end labelled primers. It is also known as thermal or linera amplification of DNA sequencing. In this 4 separate amplification reactions are set up each having same primer and different ddNTPs. 2 cycling programs are used in cycling sequencing.