Oct 22, 2009
Restriction Fragment Length Polymorphisms (RFLP’s)
-This method of analysis does not rely on the mutation having a phenotype
change, some genes can be polymorphic, therefore, this is a much more useful and
accurate method. (refer to fig 4.1 Genes IX)
-Restriction site polymorphisms change the sequence of the code and can possibly
create or destroy restriction site.
-The higher the recombination frequency the greater the distance the allele is from
the center of the gene and vice versa.
***refer to previous notes for clarification***
-Gene targeting is a useful tool for identifying the probability of phenotype
appearing (genetic diseases); it can also be used for isolating genes of interest.
Determining Gene Structure
-The first step is to identify the size of the DNA insert.
-Once the size of the insert is determined the genome of interest needs to be
mapped via restriction enzymes.
-The mapped genome can now provide the location of the gene and the actual
gene can then be analyzed by Southern Blot Analysis.
-The next step is to perform Heteroduplex mapping:
1)the isolated fragment is mixed with either sscDNA or mRNA
2)anneal so that a hybrid molecule is created
3)the hybrid is then placed on a grid and searched for heteroduplexes,
loops on the map will represent introns.
*if the hybrid does not anneal properly then it means only fragment of the
gene was isolated.
-These are uncommon occurrences but can be found everywhere
-bacteria and phages
-yeast more often than not contain introns
-higher eukaryotes have introns in most genes.
-All classes of genes may have introns (mRNA, rRNA, tRNA), mitochondrial and
chloroplast genes also contain introns.
-Related genes may have similar exon/intron arrangements, α & β globin genes
are usually familiar too.
-Pseudo-genes are non-functional genes that have the same arrangement as a wild
type gene but it is slowly decaying due to disuse. -Exon sequences are conserved but intron sequences vary over evolution. The
isolation of exons allows us to isolate and identify genes.
-Exon coding for proteins:
-ORF: open reading frames, doesn’t contain stock codons for proteins
-should be found in related animals
-Refer to previous notes
-As the chromosome walk is performed the gene needs to be tested for exons, there are a
number of ways to do so:
1)Zoo Blot – essentially a Southern Blot of genomic DNA of closely related
animals. The related genomes are then digested with restriction