2. Restriction Fragment Length Polymorphisms (RFLPs) By Amr S. Moustafa, M.D.; Ph.D. Assistant Prof. & Consultant, Medical Biochemistry Dept. College of Medicine, KSU amrsm@hotmail.com

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

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

5. 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 as Changes in DNA sequences Protein variations Diseases

6. 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

7. Polymorphisms Vs Mutations Genetic polymorphisms: Common alleles > 1% Mutations: Rare alleles < 1%

8. 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

9. 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

10. Polymorphisms: Detection – 2 At DNA level  DNA sequence analysis Inherited variations in DNA sequences Different sizes of DNA fragments Restriction enzyme  Restriction fragment length polymorphisms (RFLPs):

11. Before the Test To whom who knows the answer before Please Let me Look at Think about Answer by myself

12. The Test Madam I am Adam كل في فلك

13. ِ After the Test 5’- C T G C A G - 3’ 3’- G A C G T C - 5’ Pst I

14. RFLPs: Causes Single nucleotide polymorphisms (SNPs): Gain or loss of a restriction site more frequent than mini- & micro-satellites Variable number tandem repeat (VNTR): 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

15. RFLPs: VNTR For each person a pair of homologous chromosomes is shown

16. 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

17. RFLPs: Prenatal Diagnosis of sickle cell anemia - 1

18. RFLPs: Prenatal Diagnosis of sickle cell anemia - 2

19. RFLPs: SA Genotypes - 1 100 200 300 400 315 1 2 3 4 bp Detection of SA gene by PCR

20. RFLPs: SA Genotypes - 2 100 200 300 400 315 235 1 2 3 4 bp A1A1A2A2 Detection of PstI RFLP of SA gene by PCR