Molecular Markers: RFLP AFLP RAPD SNP Procedure - barefoot farmbooks

During the Last three decades, molecular markers have been used extensively in various genetic studies. Molecular marker is a DNA segment of known or unknown structure and function which is tightly linked with desired gene or traits.

 During 1990s and later, new markers were developed. Marker can classify broadly according to generation and protocol.

Classification based on Generation:

·         1st Generation: RFLP and RAPD

·         2nd Generation:  AFLP and SSR

·         3rd Generation: SNP

Classification Based on Protocol:

·         PCR based Marker:    RAPD

                                          AFLP

                                          SSR

·         Non PCR based Marker: RFLP

In this article I will give you a general protocol regarding RFLP, RAPD, AFLP and a small note about SNP.

RFLP (Restrictions Fragment Length Polymorphism):

The main principal of RFLP is same restrictions endonuclease produce DNA fragments of different length from same stretch of genomic DNA of different individual or different species or related species. When two or more genotype is treated in this manner any variation in the length of individual fragments among genotype will be recorded as polymorphic marker.

General protocol is -

1.    High amount of purified and intact DNA is required.

2.    Then the DNA is treated with Restrictions Endonuclease (RE)

3.    The fragments is separated through gel electrophoresis

4.    This gel is placed into alkaline solution to denatured the double stranded DNA

5.    Denatured DNA is then transferred in nitrocellulose membrane. A nitrocellulose paper is placed on the upper side of the gel. 3-4 filter papers are placed on the nitrocellulose paper. Buffer is move from beneath the gel to upper filter paper. This buffer movement transfer DNA from gel to nitrocellulose membrane. This is called blotting.

6.    Pre- hybridization buffer is used as blocking agent. Blocking reagent present in this occupy available binding site on membrane.

7.    Radioactive probe then add in nitrocellulose membrane. Probe is bind with specific base sequence present in DNA fragments.

8.    The bands to which the probe has hybridized are detected by autoradiography

9.    Any variation among band position consider as a RFLP marker

RAPD (Random Amplified Polymorphic DNA):

1.    RAPD is a PCR based marker. It denotes a genomic region that is amplified in some but not in other individual of a population when their DNA is subjected to PCR using same oligonucleotide primer.

2.    In this process 10-12 base pair long primer is needed. The primers can be designed without the experimenter having any genetic information of organisms being tested

3.    The genomic DNA of selected strain is mixed with an excess of selected primer and other reagents needed for PCR and used for DNA amplification in PCR

4.    The genomic segment contain inverted copies of the sequence complementary to the primer is amplified. Two primers should point towards each other and distance among them should be 100bp to 1500bp. The two primers should place in complementary DNA strands. If two primers placed in same DNA strands and distance among them is more than 2000bp than no amplification occur.

5.    DNA strains having complementary sequence of primer produced distinct band in agarose gel but less no of bands or no bands can observed in the DNA strain having less no of complementary sequence or no complementary sequence present

6.    Any variation among bands consider as RAPD marker.

AFLP: (Amplified Fragment Length Polymorphism)

1.    There are several protocols to conduct AFLP. So, I am sharing the basic steps to conduct AFLP.

2.    To conduct AFLP, purified and intact DNA is required. Then the DNA is divested by two different Restriction Endonuclease (RE). One of the Restriction Endonuclease is frequent cutter and other one is rare cutter.

3.    After this adapter is added to digested fragments. An adapter is a short, chemically synthesized, single or double stranded oligonucleotide having sticky ends to both or one end. It means that a specific adapter should have sticky end of specific RE. Thus adapter can ligated with DNA fragment digested by that specific RE.

4.    In AFLP two adapters is required because DNA is cut by 2 different Restriction Endonuclease. Adapter is ligated with DNA fragment having 2 different sticky ends.

5.    In this picture adapter is attached with DNA fragment having sticky ends created by EcoRI and MseI.

6.    After ligation of adapter designed primer is bind with specific site. Primer are designed to bind to following sequence --- Ligated adapter + Recognition site of Restrictions Endonuclease + 1-3 chosen base ( nucleotide base just after Recognition site)

7.    This kind of Designed primer helps to polymerization of DNA fragment.

8.    PCR is conducted after this and PCR products are run through Gel electrophoresis to obtain different banding patterns.

9.    DNA isolated from different strain of same species or different species or different genus gives different banding pattern. Selection of specific gene or traits can be done by study different banding pattern obtains from AFLP process.

 

BONUS:  SNP(Single Nucleotide Polymorphism ):

RFLP, RAPD, AFLP all are gel electrophoresis based assay. This gel based assay is costly and time consuming.  Thus new kind of molecular marker is developed which can be detected by either non-gel based assay or through microarrays. SNP represent sites where DNA sequence differs by a single base .Two or more genotype is tested and any mismatch of a single base suggests the presence of SNP

    

   Author:  

SAGNIK CHANDA
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