MOLECULAR MARKERS BASED

ON DNA

BUYEGI GEORGE

INTRODUCTION

• A genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. • With these genetic markers one is able

to distinguish from one individual to the other. These variations of individuals can be due to mutation or alteration in the genomic loci (Pourmohammad, 2014).

DNA ISOLATION

• Is a process by which scientists are able to separate/extract DNA from a cell with different substances such as protein.

Tip

•DNA can be isolated from a number of plant tissues, young plant tissues are said to be a good source of DNA, because they gradually divide and have fresh DNA compared to the older ones

DNA ISOLATION PROCEDURE

• Place a leaf tissue is d in a tube and frozen using liquid nitrogen ( freezing process helps in breaking the cell mechanically)• A mortar and pestle or a tool like drill is

used to grind the tissue into fine powder • A solution containing Buffer, Salt and

detergent is added to the tissue ( salt and buffer are to stabilize the DNA while detergent is to break the cell membrane)

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• The mixture is incubated at a 600 C, during incubation frequent mixing is required (heating disturbs the cell). • At this stage is when the contents of the

cell such as, DNA and the other cellular components are released to the solution.• A solvent such as Chloroform is added to

wash away other cellular components.

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• The sample is spun rapidly in a centrifuge. • A centrifuge forces separation of the

solvent from DNA solution. • DNA solution usually comes at the top of

the solvent. • The DNA solution is then removed using

a pipette and the solvent is poured out and test tube is cleaned.• DNA solution is placed back in the tube.

DNA PURIFICATION AND QUANTIFICATION

• DNA purification and quantification are important techniques in a way that a purified, high-quality DNA can be used in Polymarase Chain Reaction (PCR), coupled in vitro transcription/translation systems and sequencing reactions.

DNA PURIFICATION PROCEDURE• Addition of cold ethanol to the DNA

solution. The ethanol will come on top of the solution.• Mixing follows as the DNA precipitates

which are insoluble in cold ethanol comes out of the solution.• The sample is then centrifuged and the

solution is removed so that only pure DNA is remaining.

DNA QUANTIFICATION

• This is done to check the concentration and purity of DNA present in a solution mixture.• This can be done by comparing

different solutions of DNA and the one with no contamination of any components such as mitochondria or protein or any other cellular ingredient, is one that’s pure and of high quality.

METHOD OF DNA QUANTIFICATION

• Spectrophotometric method. • This is done using Nanodrop device

which is a very small spectrophotometer that can accurately read DNA concentration and purity in as little as 1μl.

DNA SEQUENCING

• This is all about finding order of nucleotides in DNA fragment.• Every strand of DNA is made up of pairs

of molecules called nucleotides strung together in a long chain called double helix. • Each pair is held together by its

complementary basis (Guanine (G), Thymine (T), Adenine (A) and Cytosine (C)). • DNA sequencing is PCR based

procedure.

DNA SEQUENCING

• Take the sample that is to be sequenced, and use PCR to amplify the sample. • PCR is able to generate millions of DNA

fragments.• During PCR addition of buffer solution

which has four free floating nucleotides, which are the A, G, C and T.• A primer is then added.

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• Then addition of Di deoxyl nucleotide (ddNTPs). • If ddNPT incorporates into the growing strand

of DNA and since it has no oxygen group it terminates elongation of the strand. • Different DNA fragments will be terminated at

different points.

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• Lastly Gel electrophoresis is used in to separate the strand by sides. • Therefore when running all the

fragments of DNA from PCR on a Gel it will separate them by sides

ADVANTAGES OF DNA SEQUENCING

• Helps plants in resisting certain diseases.• DNA sequencing allows researchers to identify changes in genes, associated with diseases and phenotypes and also identify potential drug targets.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

• Is a genetic variation that can be examined by trimming off the DNA into fragments (restriction fragments) with a restriction enzyme. •One individual can be differentiated with the other through these restriction fragments.

• Therefore a population is said to be polymorphic (having many different morphologies). •Differences may arise through mutations, base pair deletion, translocation etc.

RFLP TECHNOLOGY

•Unlike other technologies RFLP is a restriction enzyme based.• It was the first technology to be used to differentiate individuals.

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• DNA sequences are cut by using a particular restriction enzyme. • From there DNA fragments of

different lengths are obtained• Then they are separated by Agarose

gel electrophoresis. • This provides a pattern of bands

that is unique for a particular DNA being analyzed.

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• The obtained RFLPs are then denatured before southern blot; a primer is added for hybridization of the RFLPs. • After hybridization they are separated from DNA fragments.

APPLICATIONS OF RFLP

•DNA Fingerprinting.• Identification of a group of plant species at risk of certain genetic disorders.

CONCLUSION

• DNA isolation, purification and quantification are important techniques that are needed for most of DNA experiments such as PCR.• A high quality and pure DNA will

give better results in sequencing and RFLP

Thanks