Chapter 20: Genomics 1: Analysis of DNA

FISH (Fluorescence in situ hybridization)

-Chromosomes intact -Cells are treated with an agent that causes them to swell and their contents are fixed to the slide -Chromosomal DNA is denatured and DNA probe is added -Goal: determine the location of a gene within a set of chromosomes -Probe bines to a site in the chromosome where the gene is located b/c the probe and chromosomal gene line up and hydrogen bond with each other -To detect where probe is, the fluorescence is shown

To map a gene via in situ hybridization

-Researchers use a probe to detect the location of the gene within a set of chromosomes -If the gene of interest has been cloned previously, the DNA of the cloned gene can be used as a probe -Because a DNA strand from a cloned gene, which is a very small piece of DNA relative to a chromosome, hybridizes only to its complementary sequence on a particular chromosome, this technique provides the ability to localize the gene of interest

Human Genome Project

1. To obtain a genetic linkage map of the human genome 2. To obtain a physical map of the human genome 3. To obtain the DNA sequence of the entire human genome 4. To develop technology for the management of human genome information 5. To analyze the genomes of other model organisms 6. To develop programs focused on understanding and addressing the ethical, legal and social implications of the results obtained from the Human Genome Project 7. To develop technological advances in genetic methodologies

Molecular Markers

A segment of DNA found at a specific site along a chromosome and has properties that enable it to be uniquely recognized using molecular tools such as polymerase chain reaction and gel electrophoresis

Restriction fragment length polymorphism (RFLP)

A site in a genome where the distance between two restriction sites varies among different individuals. These sites are identified by restriction enzyme digestion of chromosomal DNA and the use of Southern blotting.

Microsatellite

A site in the genome that contains many short tandem repeat sequences. The total length is usually in the size range of 100-500 bp and their lengths may be polymorphic within a population. They are isolated via PCR

Southern Blotting

A technique used to detect the presence of a particular genetic sequence within a mixture of many chromosomal DNA fragments The basis for a Southern blotting experiment is that two DNA fragments bind to each other only if they have complementary sequences

Linkage mapping

Also called genetic mapping; it uses the frequency of genetic recombination between different genes to determine their relative spacing and order along a chromosome

Physical mapping

DNA cloning techniques are used to determine the location of and distance between genes and other DNA regions

High Stringency

High temperatures and/or at low salt concentrations, the probe DNA and chromosomal fragment must be very complementary--nearly a perfect match--in order to hybridize. -Close or exact complementarity between the probe and chromosomal DNA fragment

Example of Physical mapping

In a physical map, the distance are computed as the number of nucleotide base pairs between genes

Example of Linkage mapping

In eukaryotes, linkage mapping involves crosses among organisms that are heterozygous for two or more genes

How does the fluorescence happen?

It incorporates biotin-labeled nucleotides into the probe. Biotin is a small non-fluorescent molecule but has a very high affinity for a protein called avidin. When these two bind, it labels the probe

Low Stringency

Low temp and/or high salt concentrations, DNA sequences that are not perfectly complementary may hybridize to the probe. -Homologous genes with DNA sequences that are similar but not identical to the cloned gene being used as a probe

Shot-gun sequencing

Most inexpensive way. DNA fragments to be sequenced are randomly generated from larger DNA fragments -Genomic DNA is isolated and broken into smaller DNA fragments, typically 1500 bp or longer in length -The researchers isolated chromosomal DNA from H. influenzae and used sound waves to break the DNA into small fragments of approximately 2000 bp in length

Cytogenetic mapping

Relies on the localization of gene sequences within chromosomes that are viewed microscopically.

Genome-sequencing projects

Research endeavors that have the ultimate goal of determining the sequence of DNA bases of the entire genome of a given species

Hierarchical shotgun sequencing

Researchers cloned large DNA fragments into BACs o generate a contig for each chromosome. The nucelotide sequence was determined clone by clone. This strategy had the advantage of making it easier to align the overlapping sequences because the DNA sequences were grouped according to the BAC clone from which the sequence was obtained. It also minimized the amount of DNA sequencing by reducing the amount of sequencing in overlapping regions

Whole-genome shotgun sequencing

The DNA from the entire genome was isolated and sheared into small and large fragments and cloned into vectors. The sequencing of both ends (double-barrel shotgun sequencing) of longer fragments made alignment easier

Mapping

The experimental process of determining the relative locations of genes or other segments of DNA along individual chromosomes

Locus

The site within a genetic map where a specific gene or other DNA segment is found

In situ hybridization

This is widely used to cytogenetically map the locations of genes or other DNA sequences within large eukaryotic chromosomes. -The term "in situ" indicates that the procedure is conducted on chromosomes that are being held in place--adhered to a surface

Example of cytogenetic mapping

When stained, each chromosome of a given species has a characteristic banding pattern, and genes are mapped cytogenetically relative to a band location