12/1/2023
Intended Learning Outcomes
◼ What are mutations and what are
POINT MUTATIONS & their different types?
RFLP ◼ What is RFLP and what are
different methods to perform it?
◼ What are the applications and
limitations of RFLP?
MUTATION POINT MUTATIONS
◼ Any change in the genetic ◼ Occurs at 1 single point in DNA
sequence (DNA) that
affects the genetic ◼ These could cause one amino
information acid to change, which would
change the protein that is being
produced.
POINT MUTATIONS CODON TABLE
◼ BASE SUBSTITUTION:
– A nucleotide is replaced with a different
nucleotide.
– Three examples:
◼ Transversion mutation
◼ Transitionmutation
◼ Silent mutation
1
12/1/2023
BASE SUBSTITUTION
Transversion Substitution
Examples:
◼ Transversion – happens when one
purine (A,G) is swapped with a
pyrimidine (C,T).
DNA: CTT to CAT
mRNA: GAA to GUA
AA: Glu to Val
BASE SUBSTITUTION BASE SUBSTITUTION
Examples: Examples:
◼ Transition mutations – happen ◼ Silent mutations – happen when a
when one purine is swapped with the base codon is changed but both code
other purine or a pyrimidine is for the same amino acid.
swapped with a pyrimidine.
DNA: GAA to GAC
DNA: CTT to CCT
mRNA: CUU to CUG
mRNA: GAA to GGA
AA: Leu to Leu
AA: Glu to Gly
Frameshift Mutation
POINT MUTATIONS
Examples:
◼ FRAMESHIFT: Because of a point ◼ Insertion
mutation, there is a shift in the “reading – Shifts the reading to the right
frame” of genetic message
– DNA: from GCATCG to GCC ATC
– Almost always affect all the amino acids – mRNA: from CGU AGC to CGG UAG
after the mutation, which will affect the – Amino
entire protein. Acid: from Arg Ser to Arg STOP
2
12/1/2023
Point Mutation
◼ Deletion
– Shifts the reading to the left
– DNA: from GCATCG to GAT CG
– mRNA: from CGU AGC to CUA GC
– Amino
A New Subtype of Brachydactyly Type B Caused by
Acid: from Arg Ser to Leu Point Mutations in the Bone Morphogenetic Protein
Antagonist NOGGIN
RFLP
Restriction Fragment Length Polymorphism
RFLP’s contribution . . . . Introduction
• RFLP is based on DNA fragment
• Restriction fragment length polymorphism
length differences after digesting
(RFLP) markers were regarded as the first
genomic DNA with one or more restriction
shot in the genome revolution, marking
enzymes.
the start of an entirely different era in
the biological sciences. • DNA is digested by one or more
restriction enzymes and separated on an
• RFLP was the most popular approach for agarose gel.
analysis of genetic variation during the
entire 1980s. • The DNA in the gel transferred
to nitrocellulose or nylon membranes.
3
12/1/2023
• The specific DNA locus with a potential
fragment length difference is hybridized to a
probe, a radioactively labeled DNA or RNA
molecule with sequence similarities to target
• After hybridization, the nonspecific probes
must be washed away leaving only hybridized
probes to the specific locus.
• The membrane is then exposed to a piece of X-
ray film for autoradiography to visualize the
DNA bands.
Technology Advances - Development of
The obvious question . . ..
RFLP
• Two specific technological advances that
set Why the restriction enzymes do
the foundation for RFLP were —
not digest bacterial DNA ??????
– The discovery and application
of restriction enzymes &
– The development of DNA
hybridisation.
The vast nature of RE
• To date, more than 10,000 bacterial species
have been screened for the existence of
restriction enzymes; more than 3,000
restriction enzymes have been found with
more than 250 distinct specificities.
• Occasionally enzymes with novel DNA
sequence specificities are still found, but most
now prove to be duplicates (isoschizomers) of
already discovered specificities.
4
12/1/2023
The development of DNA hybridisation
The hybridization techniques are used
extensively in the research laboratory
for detecting specific nucleotide sequences
in DNA and RNA and are increasingly
being applied to detect mutations in the
candidate genes.
All of the hybridization techniques started
with a simple hybridization technique called
Southern blot (Southern 1975).
Southern blot
• A Southern blot is a method in molecular biology
for enhancing the result of an agarose gel
electrophoresis by marking specific DNA
sequences.
• The method is named after its inventor, the
British biologist Edwin Southern.
•All of these blotting techniques require the use
of molecular probes.
Probe
• Agent that is used to detect the presence of a
molecule in the sample.
• For Southern blot, the probe is a DNA sequence
that is used to detect the presence of a
complementary sequence by hybridization with a
DNA sample.
• Probes are needed to screen for a gene of
interest, to determine genomic structure and
gene copy numbers, to analyze gene expression, or
to validate allelic amplification in PCR.
5
12/1/2023
Principle and Molecular Basis of RFLP
• Restriction endonucleases cut DNA wherever
their recognition sequences are encountered
so that changes in the DNA sequence due to
indels, base substitutions, or rearrangements
involving the restriction sites can result in the
gain, loss, or relocation of a restriction site.
Two approaches are widely used for RFLP analysis Southern blot analysis
• Traditionally, fragments were separated using
Two Southern blot analysis (Southern 1975), in
which genomic DNA is
approaches – digested
– subjected to electrophoresis through an agarose gel
Use of – transferred to a membrane
hybridization Use of PCR – visualized by hybridization to specific probes.
6
12/1/2023
PCR
• In recent approaches - PCR replaces the Southern Inheritance of RFLP Markers
blot analysis.
• RFLP markers are inherited in a Mendelian
•If flanking sequences are known for a locus,
the segment containing the RFLP region is fashion and as codominant markers.-The
amplified via PCR. strength of RFLP markers.
•If the length polymorphism is caused by a • Both alleles are expressed in molecular
relatively large deletion or insertion, gel
electrophoresis of the PCR products should phenotypes (bands on gels).
reveal the size difference. •In the case of an individual heterozygous for
•If the length polymorphism is caused by base two allelic RFLP patterns on alternative
substitution at a restriction site, PCR products
must be digested with a restriction enzyme to chromosomes, the phenotype includes
reveal the RFLP. both of the patterns .
• RFLP is a non-PCR based method .
• In this Method DNA is digested with
restriction Enzymes.
• RFLP is the co-dominant marker.
In the example, a base substitution within the 8 kb fragment leads to the gain of a
new restriction site. For homozygous AA, one band of 8 kb should be • RFLP is 1-10 loci detected.
generated; for homozygous BB, two bands of 3 kb and 5 kb should be generated; for
heterozygous AB, three bands of 8 kb (from allele A), 3 kb and 5 kb (both from
allele B) should be generated.
Advantages of RFLPs
• Highly robust methodology with good
Phylogenetic Tree
transferability between laboratories.
• No sequence information required.
• highly recommended for phylogenetic analysis
between related species.
• Well suited for constructing genetic linkage
maps.
• Simplicity—given the availability of suitable
probes, the technique can readily be applied to
any animal specie.
7
12/1/2023
Disadvantages of RFLPs Applications of RFLP:
• Large amounts of DNA required. ➢RFLPs can be used in paternity cases or criminal
cases to determine the source of a DNA sample.
• Automation not possible. (i.e. it has forensic applications).
• Low levels of polymorphism in some species. ➢RFLPs can be used determine the disease status of
• Time consuming, especially with single-copy an individual. (e.g. it can be used in the detection
probes of mutations particularly known mutations).
• Costly. ➢In human population genetics, geographical
isolates and comparison of genetical makeup of
• Moderately demanding technically. related species.
THANK YOU !!!