Case details- VNTR Analysis

A 16-year-old girl was abducted when she went to a local store. Her body was found the next morning and the autopsy revealed that she had been sexually assaulted and strangled. Crime scene investigators were able to collect a semen sample from vaginal fluid as well as tissue samples from underneath the victim’s fingernails. DNA samples were obtained from three suspects besides the victim. A variable number of tandem repeats (VNTR) analysis was performed on the DNA samples from the evidence collected, the victim, and the suspect and the results were compared.

Which of the following techniques is the most appropriately applied for this analysis?

A. Thin layer chromatography

B. Polyacrylamide gel electrophoresis

C. Northern blot

D. Southern blot

E. Western blot

Details

The correct option is D- Southern Blot.

Variable numbers of tandemly repeated (VNTR) units are types of “insertions” that result in an RFLP (restriction fragment length polymorphism).

Every individual has different numbers of VNTRs. The VNTRs can be inherited, in which case they are useful in establishing genetic association with a disease in a family; or they can be unique to an individual and thus serve as a molecular fingerprint of that person.

Treatment of genomic DNA from different individuals with a single restriction enzyme does not always give the same set of fragments because some restriction sites are polymorphic, being present in some individuals but absent in others, usually because a point change in the nucleotide sequence changes the restriction site into a sequence not recognized by the restriction enzyme (figure-1).

RFLP

Figure-1- Restriction Fragment Length Polymorphism (RFLP)

Overview of Southern Blotting

Southern Blotting is named for the person who devised the technique. 

This technique involves transfer of DNA molecules from an electrophoresis gel to a nitrocellulose or nylon membrane, in such a way that the DNA banding pattern present in the gel is reproduced on the membrane. During transfer or as a result of subsequent treatment, the DNA becomes immobilized on the membrane and can be used as a substrate for hybridization analysis with labeled DNA or RNA probes that specifically target individual restriction fragments in the blotted DNA. Southern blotting is thus a method for ‘detection of a specific restriction fragment against a background of many other restriction fragments.

The steps involved in southern blotting (figure-2) are:

1) DNA is extracted from the cell.

2) It is partially digested by a restriction endonuclease (Obtaining complete fragmentation of DNA at the intended restriction enzyme sites is a critical step in Southern blot analysis).

3) The resulting DNA fragments are separated by electrophoresis and then denatured to form single-stranded molecules (ssDNA). Fragmented DNA is typically electrophoresed on an agarose gel to separate the fragments according to their molecular weights. Acrylamide gels can alternatively be used for good resolution of smaller DNA fragments (<800 bp).

4) Without altering their positions, the separated bands of ssDNA are transferred to a nitrocellulose filter and exposed to radio labeled cDNA or RNA.

5) A nucleic acid probe with sequence homologous to the target sequence under study is labeled with radioactivity, fluorescent dye, or an enzyme that can generate a chemiluminescent signal when incubated with the appropriate substrate. The probe detects complementary DNA sequences, and binds to them.

6) In the detection step, the bound, labeled probe is detected using the method required for the particular label used. For example, radio labeled probes may be detected using X-ray film or a phosphorimaging instrument, and enzymatically labeled probes are typically detected by incubating with a chemiluminescent substrate and exposing the blot to X-ray film.

Southern blot transfer

Figure-2-Southern blotting

Applications of Southern blot

Southern blotting has many applications in molecular biology, including the identification of one or more restriction fragments that contain a gene or other DNA sequence of interest and in the detection of RFLPs used in construction of genomic maps.

In the current situation (figure-3), the identity of the criminal (suspect 2) has been confirmed by this method, the electrophoretic pattern of sample DNA is similar to suspect 2.

DNA Fingerprint

Figure-3- Electrophoretic pattern of sample DNA and three suspects.

As regards other options

A. Thin layer chromatography

Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. It can also be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance.

B. Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility.

C. Northern blot

The northern blot is a technique used in molecular biology to study gene expression by detection of RNA (or isolated mRNA) in a sample. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence. They can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot.

E. Western blot

The western blot (sometimes called the protein immunoblot) is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose), where they are stained with antibodies specific to the target protein.

 

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