Download Restriction Fragment Length Polymorphisms (RFLPs)

Restriction Fragment Length
Polymorphisms (RFLPs)
By
Amr S. Moustafa, M.D.; Ph.D.
Assistant Prof. & Consultant, Medical Biochemistry Dept.
College of Medicine, KSU
[email protected]
Objectives
 Variable
Polymorphisms
RFLPs
Single nucleotide
Minisatellites
Medical
applications
number
and
Vs
tandem
polymorphisms
microsatellites
mutations
repeat (VNTR)
(SNPs)
Objectives
 Polymorphisms Vs mutations
 RFLPs
 Single nucleotide polymorphisms (SNPs)
 Minisatellites and microsatellites
 Variable number tandem repeat (VNTR)
 Medical applications
Background information: Facts
 Human DNA sequence is nearly 99.9% identical
 Only one bp varies between 2 persons
per 1500 bp DNA segment
 Each new zygote contains ~ 100 bp changes
not present in the genome of either parents
 Human genetic diversity manifested as
Changes in DNA sequences
Protein variations
Diseases
Background information: Definitions
Alleles:
Different forms of the same gene on a specific locus
Genotype:
The set of alleles that make up the genetic constitution
Phenotype:
The observable expression of a genotype
Polymorphisms Vs Mutations
Genetic polymorphisms:
Common alleles > 1%
Mutations:
Rare alleles
< 1%
Polymorphisms: Sites
Inter-genes or intronic:
Detected by DNA sequence analysis
Gene coding sequences:
Different protein variants
Distinct phenotypes (may be)
DNA regulatory regions:
may affect phenotypes
Polymorphisms: Detection – 1
At protein level
ABO System:
A, B & O alleles: Chromosome 9
A & B: 4 nucleotide differences
O: One base deletion and frame-shift
Phenotypes: O, A, B & AB
Rh System:
Rh-D: Chromosome 1
Phenotypes: Rh-positive and Rh-negative
Incompatibility:Hemolytic disease of newborn
Polymorphisms: Detection – 2
At DNA level
 DNA sequence analysis
 Restriction fragment length polymorphisms
(RFLPs):
Inherited variations in DNA sequences
Restriction enzyme
Different sizes of DNA fragments
Before the Test
To whom who knows the answer before
Please
Let me
Look at
Think about
Answer by myself
The Test
Madam I am Adam
‫كل في فلك‬
Madam I am Adam
‫كل في فلك‬
ِAfter the Test
5’- C T G C A G - 3’
3’- G A C G T C - 5’
Pst I
RFLPs: Causes
Single nucleotide polymorphisms (SNPs):
Gain or loss of a restriction site
Variable
repeat
(VNTR):
morenumber
frequent tandem
than mini& micro-satellites
Alteration of number of nucleotides
between restriction sites:
Minisatellites: 10 - 100 bp
Microsatellites: 2 - 4 bp
2 unrelated individuals: different pattern
Identical twins: identical pattern
RFLPs: VNTR
For each person a pair of
homologous chromosomes
is shown
RFLPs: Medical applications
 Mapping a gene to a particular region
of a chromosome
 Tissue typing for organ transplantation
 Paternity testing and forensic applications
 Prenatal diagnosis of genetic diseases
RFLPs: Prenatal Diagnosis of
sickle cell anemia - 1
RFLPs: Prenatal Diagnosis of
sickle cell anemia - 2
RFLPs: SA Genotypes - 1
bp
1
2
3
4
400
300
315
200
100
Detection of SA gene by PCR
RFLPs: SA Genotypes - 2
bp
1
2
3
4
400
315
300
235
200
100
A1 A1A2 A2
Detection of PstI RFLP of SA gene by PCR