Restriction Fragment Length

Polymorphisms (RFLPs)

By

Amr S. Moustafa, M.D.; Ph.D.

Assistant Prof. & Consultant, Medical Biochemistry Dept.

College of Medicine, KSUamrsm@hotmail.com

Objectives

Polymorphisms Vs mutations RFLPs Single nucleotide polymorphisms (SNPs) Minisatellites and microsatellites Variable number tandem repeat (VNTR) Medical applications

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

Each new zygote contains ~ 100 bp changes not present in the genome of either parents

Only one bp varies between 2 persons per 1500 bp DNA segment

Human genetic diversity manifested asChanges in DNA sequencesProtein variationsDiseases

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 variantsDistinct phenotypes (may be)

DNA regulatory regions:may affect phenotypes

Polymorphisms: Detection – 1

At protein levelABO System:

A, B & O alleles: Chromosome 9A & B: 4 nucleotide differencesO: One base deletion and frame-shiftPhenotypes: O, A, B & AB

Rh System:Rh-D: Chromosome 1Phenotypes: Rh-positive and Rh-negativeIncompatibility:Hemolytic disease of newborn

Polymorphisms: Detection – 2At DNA level

DNA sequence analysis

Inherited variations in DNA sequences

Different sizes of DNA fragments

Restriction enzyme

Restriction fragment length polymorphisms (RFLPs):

Before the Test

To whom who knows the answer before

Please

Let meLook atThink aboutAnswer by myself

The Test

Madam I am Adam Madam I am Adam

فلك في كل فلك في كل

;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 sitemore frequent than mini- & micro-satellitesVariable number tandem repeat (VNTR):Alteration of number of nucleotides between restriction sites:

Minisatellites: 10 - 100 bpMicrosatellites: 2 - 4 bp

2 unrelated individuals: different patternIdentical twins: identical pattern

RFLPs: VNTR

For each person a pair ofhomologous 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

100

200

300

400

315

1 2 3 4bp

Detection of SA gene by PCR

RFLPs: SA Genotypes - 2

100

200

300

400

315

235

1 2 3 4bp

A1 A1A2 A2

Detection of PstI RFLP of SA gene by PCR