Question

a)What are molecular beacons and how can they be useful in performing multiple assays simultaneously?
b)How is DNA replicated?
c)How is a DNA (oligonucleotide) microarray manufactured?

Answer 1

a. Molecular beacons are single stranded hairpin oligonucleotide probes used in real time PCR. When they are encounter target DNA, they will unfold, bind and then fluoresce, which is then detected. They have a stem loop structure. The loop is 18-30 bp region, which is complementary to target DNA. The stem is 5-7 bp long present at end of loop, with complementary ends. The 5’ end has a 5’ fluorophore, which will fluoresce when beacon binds target DNA. The 3’ fluorophore present at 3’ end is the quencher, that prevent fluorescence when the beacon is in stem loop structure.

Molecular beacons can be used for multiplexing as they can be tagged with different fluorescent dyes with a common quencher. They also have thermodynamic properties for specific detection of different nucleic acids based on the complementary loop structure. In multiplex qPCR, different genes are simultaneously detected in same tube. When the beacon is not bound to DNA, the quencher will prevent fluorescent signals being emitted. However, when the target DNA is present, the beacon will unfold, the loop binds to target DNA and 5’ fluorophore will release the signal, which is detected. As different fluorescence dyes are used as 5’ fluorophores for different genes, the amplification can be detected specific for each target gene.

b. DNA is replicated by a process of DNA replication to form daughter DNA which is identical to the parent DNA. The mode of DNA replication is semiconservative, in which each parental DNA strand acts as a template for DNA synthesis. The DNA helicase will first unwind the DNA, by breaking the hydrogen bonds between DNA nucleotides. The topoisomerase will prevent negative supercoiling. Primase synthesizes DNA primers, which binds to DNA. Primers are used for lagging strand synthesis but not leading strand synthesis. DNA polymerase will add nucleotides to the 3’ OH group of the growing strand. Direction of DNA synthesis is 5’ to 3’ direction. DNA ligase will seal the gaps in leading strands synthesized. The exonuclease and proofreading activity of DNA polymerase is involved in DNA repair in cases incorrect bases are incorporated in DNA.

c. Microarrays are oligonucleotides arrays where in oligonucleotide probes are attached to a matrix. The matrix can be glass or nylon or silica. The oligonucleotides probes are short single stranded sequences complementary to DNA sequences of different genes. The oligonucleotide probes are tagged with fluorescent dyes. DNA microarrays can be prepared by in situ DNA synthesis by photolithography. Here, masks are used to direct oligonucleotide synthesis. Inkjet printing mediated method and semiconductor directed synthesis are other methods used for microarray preparation. In inkjet method, expel small droplets of probes on the glass slides. The probes are first PCR amplified, and then spotted.

Spotted microarrays will involve spotting the oligonucleotides to glass plate. The probes used are short chemically synthesized sequences of 25-80 bp. The probes should lack palindromic sequences and have comparable melting temperatures within 5⁰C. They have modified 5’ or 3’ ends which help in attachment to glass surface.

Oligonucleotides can be generated from libraries by cleavage and PCR. These library arrays can cover entire transcriptomes.

Insitu oligonucleotide arrays also use oligonucleotides attached to 1.2 cm2 quartz water. However, the probes are 20-25 nucleotides. Multiple probes, around 11 probes per 600 bases, are used. In Gene chips, semiconductor-based photochemical synthesis is used to synthesize the probes. The microarray surface is protected from nucleotide binding till UV light is exposed. The reactive nucleotide with photolabile protecting group will then attach to the surface of matrix. This is the photolithography mask method.

High density arrays have beads that are attached to 29 nucleotide address which is the attached to probe. This probe will bind to biotinylated cRNA probes. Each gene will have two probes sequences. Microarrays are also designed where the primers detect the 3’ end of the poly A tail.