Download Restriction Mapping Restriction Fragment Length Polymorphism

Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Restriction Mapping
DNA is restriction digested with restriction
enzymes, individually (single-enzyme digest)
and in combination (double digest).
The restriction fragments are subjected to
electrophoresis.
The fragments are identified, either using UV
absorbing dye or labeled probe.
Double digest determines if fragment produced
by one enzyme contains restriction sites for
the other enzyme.
Fragments are aligned by size.
Enzyme 1: 8 kb, 6 kb, 3 kb or 3 kb, 6 kb, 8 kb
6 kb, 8 kb, 3 kb or 3 kb, 8 kb, 6 kb
8 kb, 3 kb, 6 kb or 6 kb, 3 kb, 8 kb
Enzyme 2: 10 kb, 7 kb or 7 kb, 10 kb
Double Digest: 3 kb fragment is split into
2 kb and 1 kb fragments.
Restriction Fragment Length Polymorphism (RFLP)
Individuals can be identified according to RFLP
genotype. RFLP locus could be linked to a gene, and
thus be used as a diagnostic marker.
Use of Restriction Fragment Length Polymorphism
I.
Marker locus
II.
Diagnostic
A. Medicine
B. Forensics
III.
Assessment of Genetic Variation
A. Within and between populations
B. Within and between species
Restriction Mapping versus RFLP Mapping
I. Restriction Mapping
A. Based on physical analysis of DNA
B. Based on restriction sites with no variation
C. Mostly short-range (fine-scale) maps
II. RFLP Mapping
A. Based on recombination analysis of matings
B. Based on restriction-site variation between
homologous chromosomes
C. Mostly longe-range (coarse-scale) maps
Other Useful Approaches
1. Single Nucleotide Polymorphisms (SNPs)
Individuals differ in single nucleotides (every 11 to
300 bp in interval).
2. Simple-Sequence Length Polymorphisms (SSLPs)
Very short repetitive DNA sequences are more
polymorphic than RFLP sequences. These are
also called Variable Number Tandem Repeats
(VNTRs)
- Minisatellite Markers
- Microsatellite Markers
Simple-Sequence Length Polymorphisms
1. Minisatellite DNA
These are 1 to 5 kb in length consisting of repeats
15 to 100 nucleotides in length and are identified
by Southern analysis.
2. Microsatellite DNA
These are tandem repeats of dinucleotides,
commonly stretches of CA.
5’ C A C A C A C A C A C A C A 3’
3’ G T G T G T G T G T G T G T 5’
These are identified by gel electrophoresis of PCR
products.
Refer to Figure 4-15, Griffiths et al., 2015.
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Dideoxy DNA Sequencing
Chain-terminating
(dideoxy) nucleotide
Use of dideoxy nucleotide
in primer extension
reaction will randomly
arrest DNA synthesis.
Dideoxy DNA Sequencing
4 different reactions are
conducted, each with a
different type of dideoxy
nucleotide.
Fragments are separated
by electrophoresis.
Banding pattern is used
to infer the base
sequence of the original
template strand.
Migration
3’
Sequencing
Gel Using
Radioactive
Primer
5’
Remember…
This is base
sequence of
synthesized
strand.
Reading the DNA sequence from an automatic sequencer
Oligonucleotide primers can be tagged with fluorescent dyes
instead of radioactive labels.
A different colored dye can be used for each of the four reactions.
In Search of Potential Genes
Open reading frames (ORFs) are long stretches of DNA
that start with ATG and end with a stop codon. A doublestranded DNA molecule has 6 possible reading frames, 3
for each strand.