Tools of Human Molecular Genetics

ANALYSIS OF INDIVIDUAL DNA AND RNA SEQUENCES

Two fundamental obstacles to carrying out their investigations of the molecular basis of hereditary disease:

1- obtaining a sufficient quantity of a DNA or RNA sequence of interest to allow it to be analyzed, because each cell generally has only two copies of a gene and some genes may be transcribed only in a subset of tis­sues or only at low levels, or both, providing only a small number of messenger RNA (mRNA) molecule.

2- purifying the sequence of interest from all the other segments of DNA or mRNA molecules present in the cell.

technological revolution that solved both the problems of quantity and of purification. These two complementary technologies are molecular cloning and the polymerase chain reaction (PCR)

Molecular Cloning

Restriction Enzymes

DNA ligase­

Vectors

A vector is a DNA molecule that can replicate autonomously in a host such as bacterial or yeast cells.

The ability to generate any desired number ofidentical copies (clones) of a particular sequence

is a product of recombinant DNA technology .

Polymerase­Chain­Reaction­(PCR)Polymerase­Chain­Reaction­(PCR)An alternative method to cloning for generating essentially An alternative method to cloning for generating essentially

unlimited amounts of a sequence of interestunlimited amounts of a sequence of interest

METHODS OF NUCLEIC ACID ANALYSIS

Several techniques are available.

Depends on the size of the DNA molecule involved:

1- Small molecules, such as normal and recombinant versions of plasmids and phage chromosomes.

2- large DNA molecules contained in eukaryotic chromosomes.

Nucleic Acid Probes

Identification­Methods

- It is usually easier to identify directly the correct recombinant DNA molecule. This can be achieved by the important technique of hybridization probing

Probe labelling1- Traditionally the probe is labelled with a radioactive nucleotide,

2- non-radioactive

Radioactive labelling methods are starting to fall out of favour, however, partly because of the hazard to the researcher and partly because of the problems associated with disposal of radioactive waste. The hybridization probe may therefore be labelled in, a non-radioactive manner.

A number of methods have been developed:

a- Biotin

b- peroxidase

­­­­­­­­­­­­­­­­­­­­­­­­­­­­Kinds of blotting­

1- Southern transfer:

Transfer of DNA bands from an agarose gel to a membrane makes use of the technique perfected in 1975 by Professor E.M. Southern and referred to as Southern transfer.

2- northern' transfer:used for the transfer of RNA molecules­.

3- western' transfer:used for the transfer of protein.

- Southern transfer and hybridization can be used to locate the position of a cloned gene, or one isolated by PCR, within any DNA molecule for which a restriction map has been obtained.

- Southern hybridization used to determine the exact position of a gene within the cloned fragment.

- Also the cloned fragment may carry a number of genes in addition to the one under study.

- Southern analysis of the recombinant DNA molecule to locate the precise position within the cloned DNA fragment of the gene being sought .

Southern Blotting

Northern hybridization

Locating the position of a gene on a largeDNA molecule

-Southern transfer procedure is appropriate for most plasmids, bacteriophages and viruses, but cannot be used to locate genes on larger DNA molecules.

- Restriction mapping becomes very complicated with molecules more than about 250 kb in size, as can beappreciated by referring back to Figure 4.18.

-Imagine how much more complicated the analysis would be if there were five times as many restriction sites.

- Other techniques must therefore be used to locatethe positions of eukaryotic genes on chromosomal DNA molecules.

In situ hybridization to visualize the position of a gene on a eukaryotic chromosome

The much larger molecules (>50000kb) of mammals and other highereukaryotes are still some way beyond the capability of the current technol-ogy. Gene location on these larger DNA molecules can ,however, be achievedby in situ hybridization, which has the added advantage :

1- Not only identifying which chromosome a gene lies on,

2-but also providing information on the position of the gene within its chromosome.

In situ hybridization derives from the standard light microscopy tech-niques used to observe chromosomes in cells

In situ hybridization provides a direct visual localization of acloned gene on the light microscopic image of a chromosome.

Cells are treated with a fixative, attached to a glass slide. and thenincubated with ribonuclease and sodium hydroxide to degrade RNA anddenature the DNA molecules. Base pairing between the individual: poly-nucleotide strands is broken down, and the chromosomes unpack to a certainextent,. exposing segments of DNA normally enclosed within their strture

in situ hybridization with radioactively labelled probes has been used to position a number of genes on the human cytogenetic map.

As an alternative to radioactive labelling, a fluorescent marker can be attached to the probe and hybridization observed directly, using a special type of light microscope.

This technique, fluorescence in situ hybridization (FISH), is also frequently used with probes whose normal chromosomal locations are already known. This is particularly useful for studying cells in which chromosomal rearrangements have occurred.

DNA sequencing -working out thestructure of a gene

Probably the most important technique available to the molecular biologist is DNA sequencing, by which the precise order of nucleotides in a piece of DNA can be determined. DNA sequencing methods have been around for 35 years, but only since the late 1970s has rapid and efficient sequencing been possible.

Two different techniques were developed:

1- The chain termination method by F. Sanger and A.R. Coulson in the UK,

2- the chemical degradation method by A. Maxam and W. Gilbert in the USA.

- The two techniques are radically different but equally valuable. Both allow DNA sequences of several kilobases in length to be determined in the minimum of time.

- The DNA sequence is now the first and most basic type of information to be obtained about a cloned gene.

Automated DNA sequencing