Polymorphism & Restriction Fragment Length

Polymorphism(RFLP)

Genome Variation

• Definition:– In 2 non-related individuals: 0.1% genome

variation (1 in each 1500 bases)

Polymorphism MutationClinical effect Clinically harmless (No effect

on phenotypePotentially harmful (results in genetic disease

incidence ≥ 0.1% rare: 0.1%

Polymorphism

• Polymorphism: is a variation in nucleotide sequence from one individual to another, occurring in the non-coding regions of DNA.

• RFLP: is a genetic variant that can be examined by cleaving the DNA into fragments (Restriction fragments) with a RE. The length of the restriction fragments is altered if the genetic variant alters the DNA so as to create or abolish a site of RE cleavage. It can be used to detect human genetic variations, e.g. in prospective parents or in fetal tissue

• 2 types of DNA variation result in RFLP:

• SNP• VNTR

Considered markers, which, in most cases, have NO known effect on the structure or rate of production of any particular protein.

Single Nucleotide Polymorphism (SNP; 90% of human genome variation)

Variable Number of Tandem Repeats (VNTR)

• Tandem repeats:– short sequences of DNA at scattered

locations in the genome– Repeated in tandem (one after another)– The # of these units varies from person to

person, but is unique for any given individual– Serves as a molecular fingerprint

Variable Number of Tandem Repeats (VNTR)

VNTR loci: sites in genome that are frequently showing VNTR, important in DNA fingerprinting analysis (e.g. in forensic & paternity identity)

RFLP of VNTR

Prenatal Diagnosis• It is recommended if there is a history of severe genetic disease,

(affected previous child or near relative)

• Its aim is to determine the presence of the disorder in a developing fetus

• Methods available:1. Visualization of the fetus e.g. by Ultrasound or fetoscopy:

– If the genetic abnormality → gross anatomic defects e.g. neural tube defect

2. Amniotic fluid biochemical analysis: – e.g. level of a fetoprotein (increase in open neural tube defects, & low in

Down syndrome.– Fetal cells in amniotic fluid or in chrorionic villi biopsy: Karyotyping: the

morphology of the metaphase chromosomes (e.g trisomies, translocations →abnormal length of chromosome

3. Fetal DNA analysis: (the most detailed genetic picture; DNA from WBC, Amniotic fluid, or Chorionic villi– DNA has to be amplified first:

• In the past: cell culture• Now: PCR

Examples of diseases diagnosed prenatally

Sickle cell anemia: Could be diagnosed prenatally by:

I- Electrophoresis analysis of the amount & type of Hb obtained by hemolyzing fetal blood

1- risk in obtaining fetal blood2- Late detection (2nd trimester)

2- Fetal DNA then do RFLP: Advantages: 1- Safe2- early detection

RFLP in a family with a child affected by PKU