Dr. Riddhi Datta

Genetic marker

• A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species.

​

• It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be observed.

​

• Genetic markers can of several types:
• RFLP(Restriction fragment length polymorphism)
• AFLP (Amplified fragment length polymorphism)
• RAPD(Rapid amplified polymorphic DNA)
• STR (Short tandem repeats
• ISSR(Inter simple sequence repeat)
• SCAR (sequence characterized amplified region)
• EST (Expressed sequence tag)
• SNP(single nucleotide polymorphism
• SSR(simple sequence repeat)

​

Dr. Riddhi Datta

Restriction Fragment Length Polymorphism (RFLP)

• Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases.

​

• RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination.

​

• Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific.

​

• An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus.

​

• Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes.

​

• The RFLP probes are frequently used in genome mapping and in variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.).

Dr. Riddhi Datta

The traditional method of the RFLP is the blotting-based probe hybridization method and exploits Southern blot technique. 

Dr. Riddhi Datta

The PCR-based RFLP method is a newer version, easy and rapid.  This technique is also known as Cleaved Amplified Polymorphic Sequences (CAPS).

�

Dr. Riddhi Datta

Dr. Riddhi Datta

Amplified fragment length polymorphism (AFLP)

• Amplified Fragment Length Polymorphisms (AFLPs) are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites.

​

• The AFLP technique is based on the selective PCR amplification of restriction fragments from a total RE digest of genomic DNA.

​

Advantages: 

• No prior sequence information is required in the AFLP. 
• Also, the sensitivity, reproducibility and resolution of the AFLP marker are higher as compared with other markers. 

​

Disadvantages: 

• The AFLP marker is not co-dominant hence it can not distinguish homozygous from heterozygous. 

​

Dr. Riddhi Datta

1. Template fragments are generated by digestion of genomic DNA with two REs (EcoRI and MseI). Blue and red arrows represent EcoRI and MseI restriction enzyme sites, respectively.

2. Ligation of the double-stranded EcoRI- and MseI-specific adapters to the fragment ends.

3. A pre-amplification step using primers that match the adapter sequences and that carry each one selective nucleotide (represented by N) at their 3’ end are used to PCR-amplify subsets of the EcoRI/MseI templates.

Dr. Riddhi Datta

4. A final selective PCR-amplification step in which additional selective nucleotides are added to the EcoRI and MseI primers

​

5. The electrophoretic size fractionation and the display on denaturing polyacrylamide gels of the EcoRI/MseI amplification products.

Dr. Riddhi Datta

DNA Fingerprinting

• DNA fingerprinting or DNA profiling is a process used to determine the nucleotide sequence at a certain part of the DNA that is unique in all human beings.

​

• The DNA of every human being on the planet is 99.9% same. However, about 0.1% or 3 x 10⁶ base pairs (out of 3 x 10⁹ bp) of DNA is unique in every individual.

​

• Human genome pos­sesses numerous small non-coding but inheritable bases sequences which are repeated many times. They do not code for proteins but make-up 95% of our genetic DNA and usually possess regulatory function.

​

• They can be separated as satellite from the bulk DNA during density gradient centrifugation and hence called satellite DNA.

​

• In satellite DNA, repetition of bases is in tandem. Depending upon length, base composition and numbers of tandemly re­petitive units, satellite DNAs have subcat­egories like microsatellites and mini-satellites.

Dr. Riddhi Datta

DNA Fingerprinting

• Satellite DNAs show poly­morphism (variation at genetic level arises due to mutations).

​

• Variations occur due to mutations. These mutations in the non-coding sequences have piled up with time and form the basis of DNA polymorphism.

​

• At specific loci on the chromosome the number of tandem repeats varies between individuals. There will be a certain number of repeats for any specific loci on the chromosome.

​

• Depending on the size of the repeat, the repeat regions are classified into two groups:
• Short tandem repeats (STRs) that contain 2-5 base pair repeats
• Variable number of tandem repeats (VNTRs) that have repeats of 9-80 base pairs

​

• Since a child receive 50% of the DNA from its father and the other 50% from his mother, so the number VNTRs at a particular area of the DNA of the child will be different may be due to insertion, deletion or mutation in the base pairs.

​

• As a result, every individual has a distinct composition of VNTRs and this is the main principle of DNA fingerprinting.

Dr. Riddhi Datta

DNA Fingerprinting

• A single change in nucleotide may make a few more cleavage site of a given nucleotide or might abolish some existing cleavage site.

​

• Thus, if DNA of any individual is digested with a restriction enzyme, fragments pattern (sizes) will be produced and will be different in cleavage site position.

​

• This is the basics of DNA fingerprinting.

​

• Restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) amplification of short tandem repeats (STRs) are two main DNA tests widely used for DNA fingerprinting.

​

Dr. Riddhi Datta

Dr. Riddhi Datta

Applications of DNA Fingerprinting

• Forensic Science:

Biological materials used for DNA profiling are: Blood, Hair, Saliva, Semen, Body tissue cells etc. DNA isolated from the evidence sample can be compared through VNTR prototype. It is useful in solving crimes like murder and rape.

​

• Paternity and Maternity Determination:

A Person accedes to his or her VNTRs from his or her parents. Parent-child VNTR prototype analysis has been used to solve disputed cases, inheritance cases, immigration cases.

​

• Personal Identification:

It utilizes the concept of using DNA fingerprints as a sort of genetic bar code to pinpoint individuals.

​

• Diagnosis of Inherited Disorders:

It is also useful in diagnosing inherited disorders in both prenatal and newborn babies like cystic fibrosis, hemophilia, Huntington’s disease, familial Alzheimer’s, sickle cell anemia, thalassemia, etc.

​

• Development of Cures for Inherited Disorders:

By studying the DNA fingerprints of relatives who have a history of some particular disorder, DNA prototypes associated with the disease can be ascertained.

​

• Detection of AIDS:

By comparing the band of HIV “RNA” (converted to DNA using RTPCR) with the bands form by the man’s blood, person suffering with AIDS can be identified.