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Chapter 15

55-211 Chapter Notes - Chapter 15: Chromosome, Cloning, Factor Viii

Biological Sciences
Course Code
BIOL 2111

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Chapter 15 Genome Wide Analysis of Genetic Variation
15.1 Genetic Variation Among Individual Genomes
Extensive Allelic Variation Distinguishes Individuals within A Species
A locus with 2 or more alleles that are each present in more than 1 percent of a species’
members is considered to be a polymorphic, and the alleles of a polymorphic locus are
called genetic variations, rather than wild type or mutant
There is no such thing as a wild type human genome length
Polymorphic deletions, insertions, and duplications result in genome lengths that differ
by as much as 1 percent in healthy individuals
A locus is now considered to be any location in the genome that is defined by
chromosomal coordinates for the convenience of researchers, irrespective of biological
A DNA locus can contain multiple genes or no genes, it can be a single base pair or
millions of base pairs, as long as it has a defined genomic location and length
Since a locus can be any defined segment of DNA in the genome, an allele of the locus is
any variation in the DNA sequence itself, even if it has no impact on the expression of
any trait
Whether it is functional or not makes no difference in the manner that a locus is
transmitted from one generation to the next
Genetic Variants are Classified According to Several Criteria
Simplest and most generally useful class of genetic variants are the single nucleotide
polymorphisms, SNPS
SNPs are particular base positions in the genome where alternative letters of the DNA
alphabet commonly distinguish some people from others
Beyond the first category of SNPs, genetic variants arise in every size and complexity
1. Short deletions and insertions called InDels or DIPs
2. Regions of repeating 2 or 3 base long units termed simple sequence repeats
3. Large regions of duplication or deletion (copy number polymorphisms, CNPs, or
copy number variants, CNVs, depending on their frequency of occurrence)
4. A catch all category of complex variants that do not fit into any other category
15.2 SNPs and Small Scale Length Variations
Simplest type of DNA polymorphism is the single base SNP, which arises form a rare
mistake in replication or due to a mutagenic chemical
The Origin of Hyman SNPs is Determined by Comparison with other Species
SNP distributions
Although SNPs in coding sequences can alter the amino acid
sequence of a gene product and have a direct impact on phenotype
Vast majority of SNPs appear to be functionally silent
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Particular region is denser than average in transcription units with
3 functional genes that include the cystic fibrosis transmembrane
receptor (CFTR), which can mutate to cause cystic fibrosis
No evolutionary advantage or disadvantage is present for
mutations at these non-coding, non-regulatory loci
Single base mutations that occur at non-functional sites will not
be selected against, and although most are lost just by chance,
some will remain and gain frequency in a population
Functional SNPs will be subject to selective pressures like other
functional mutations
Human SNPs
Most SNP variation among people is confined to a limited number
of positions
Figure 15.3 is a display of the SNP differences observed in a
comparison of the personal genomes of Watson or Venter against
the genome represented in the human reference sequence
In some large blocks of genome differences of either man with the
reference genome are sparse and unique
In these blocks, Watson and venter are no more related to each
other than either is to the reference sequence
Block patterns of SNP similarity and dissimilarity provide the
foundation for genome wide associations studies
Some SNPs that do not have a direct effect on phenotype lie so
close to a disease gene, or other genes influencing significant
phenotypic differences, that they can serve as DNA markers:
specific DNA loci with identifiable variations
Medical researchers can use such markers to identify and follow
phenotypic differences in groups of people
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SNPs Can be Genotyped with Several Difference Molecular Methods
Because alleles of a SNP locus are well defined single base changes in DNA sequence,
they can be distinguished by a variety of molecular biology protocols that operate upon,
or resolve, specific DNA sequences
o Include restriction enzyme digestion, gel electrophoresis, southern blotting, PCR,
allele specific oligonucleotide hybridization, and DNA microarrays
Southern Blot
Analysis of
Restriction Site
Altering SNPS
Genomic DNA from the test smaples is treated with EcoRI and
the digested DNA separated by gel electrophoresis
Resulting southern blot is hybridized with a DNA probe
obtained from the region between the polymorphic restriction
site and an adjacent non-polymorphic restriction site
PCR Analysis of
Restriction Site
Altering SNPs
Protocol has 3 steps:
1. Amplification by PCR of a several hundred base pair
region encompassing the SNP
2. Exposure of the PCR products to the appropriate
restriction enzyme
3. Evaluation of the samples by gel electrophoresis and
ethidium bromide staining, followed by a reading of the
size of the DNA fragments off the gel
Sickle cell anaemia occurs, as we have seen, when a person
carries 2 copies of a mutant form of the HBB gene with a single
base substitution that replaces an A with a T and changes the
encoded amino acid from glutamic acid to valine
The normal allele is called A, and the sickle cell one is called S
Since the sickle cell mutation also by chance destroys the
recognition site of the restriction enzyme MstII, it is possible to
use PCR and restriction enzyme digestion to detect the mutant
Detection of any
SNP with Allele
Most SNP variants do not alter restriction sites
Only with very short probes, oligonucleotides containing
around 4o bases can single base changes provide a large
enough difference to be readily detected
Reason is that for very small DNA molecules, those composed
of no more than 60 bp, the length of the molecule itself helps
determine whether the double helix remains intact or falls apart
Effective length, and therefore the strength of the hydrogen
bond forces holding together the double helix of a short probe/
short target DNA hybrid, depends on the longest stretch that
does not contain any mismatches
When the 2 strands do not match exactly, there may not be
enough weak hydrogen bonds in a row to hold them together
Once a critical number of hydrogen bonds required for double
helix stability is achieved, any further increase in the number of
these bonds makes no difference
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