molecular markers22 (1).pptx documentation

Molecular biology Molecular markers are identifiable and measurable characteristics of DNA sequences or proteins that can serve as indicators for various biological processes, traits, or conditions. These markers are used extensively in research, diagnostics, and genetic studies. Here are some key points describing molecular markers:

1. **Definition**: Molecular markers are specific DNA sequences or proteins that can be detected and measured in a biological sample.

2. **Types**: There are several types of molecular markers, including:
- **SNPs (Single Nucleotide Polymorphisms)**: Variations at a single nucleotide position in the genome.
- **Microsatellites (Simple Sequence Repeats)**: Short, repetitive DNA sequences that vary in length among individuals.
- **VNTRs (Variable Number Tandem Repeats)**: Longer repetitive sequences where the number of repeats varies among individuals.

3. **Applications**:
- **Genetic Diversity**: Used to assess genetic variability within and between populations.
- **Disease Susceptibility**: Linked to specific diseases or conditions based on association studies.
- **Forensic Identification**: Used in forensic science for DNA profiling and identification.
- **Phylogenetics**: Used in evolutionary biology to study relationships between species or populations.
- **Marker-assisted Selection**: Used in plant and animal breeding to select for desired traits.

4. **Detection**: Molecular markers are detected using various techniques such as PCR (Polymerase Chain Reaction), DNA sequencing, gel electrophoresis, and microarray analysis.

5. **Advantages**: They provide a precise and objective way to study genetic variations and relationships without relying solely on observable traits.

6. **Limitations**: Some markers may not be directly linked to the trait of interest (they might be in linkage disequilibrium), and markers can sometimes show variability due to environmental factors.

Overall, molecular markers are crucial tools in modern biology, allowing researchers to explore genetic diversity, understand disease mechanisms, and enhance breeding and selection programs in agriculture and medicine.

1. PCR, RLFP, RAPDs

2. In biology and medicine, a molecular marker can be a substance native to the organism whose detection indicates a particular disease state (for example, the presence of an antibody may indicate an infection). In genetics, a molecular marker (genetic marker) is a fragment of DNA sequence that is associated to a part of the genome. Different kinds of molecular markers exist, such as restriction fragment length polymorphisms ( RFLPs ) , RAPDs , AFLPs , microsatellites and SNPs . They differ in a variety of ways: Their technical requirement time /money /labour needed the number of genetic markers that can be detected throughout the genome The amount of genetic variation found at each marker in a given population. MOLECULAR MARKERS

3. Overview of major marker systems The polymerase chain reaction ( PCR ) is a technique widely used in molecular biology used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA generated is used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. b) A restriction fragment length polymorphism is a variation in the DNA sequence of a genome that can be detected by breaking the DNA into pieces with restriction enzymes and analyzing the size of the resulting fragments by gel electrophoresis. It is the sequence that makes DNA from different sources different, and RFLP analysis is a technique that can identify some differences in sequence (when they occur in a restriction site).

4. Overview of major marker systems cont’ c) Random Amplified Polymorphic DNA markers (RAPDs) were first described in 1990. They are detected using the polymerase chain reaction (PCR), a widespread molecular biology procedure allowing the production of multiple copies (amplification) of specific DNA sequences. The analysis for RAPD markers is quick and simple, although results are sensitive to laboratory conditions. d) Amplified Fragment Length Polymorphism (AFLPs) markers. PCR-based method of generating molecular markers. With this technique, the DNA treated with restriction enzymes is amplified with PCR. It allows selective amplification of restriction fragments giving rise to large numbers of useful markers which can be located on the genome relatively quickly and reliably. Unlike other methods described here, the technique is patented.

5. Overview of major marker systems cont’ e ) Microsatellites: also known as Simple Sequence Repeats (SSRs) are simple DNA sequences (e.g. AC), usually 2 or 3 bases long, repeated a variable number of times in tandem. They are easy to detect with PCR and a typical microsatellite marker has more variants than those from other marker systems. Initial identification of microsatellites is time-consuming. f) SNPs: in recent years, Single Nucleotide Polymorphisms (SNPs), i.e. single base changes in DNA sequence, have become an increasingly important class of molecular marker. The potential number of SNP markers is very high, meaning that it should be possible to find them in all parts of the genome, and micro-array procedures have been developed for automatically scoring hundreds of SNP loci simultaneously at a low cost per sample.

6. Polymerase Chain Reaction ( PCR ) Developed in 1984 by Kary Mullis “Molecular photocopying" The polymerase chain reaction is a test tube system for DNA replication that allows a "target" DNA sequence to be selectively amplified, or enriched, several million-fold in just a few hours. PCR applications employ a heat-stable DNA polymerase, mostly Taq polymerase , an enzyme originally isolated from the bacterium Thermus aquaticus . This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as the template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

7. Principles and Procedures of PCR PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. A basic PCR set up requires the following components and reagents: DNA template that contains the DNA region (target) to be amplified. Two primers , which are complementary to the DNA regions at the 5' (five prime) or 3‘ (three prime) ends of the DNA region. Taq polymerase with a temperature optimum at around 70°C. Deoxynucleotide triphosphates (dNTPs), the building blocks from which the DNA polymerases synthesizes a new DNA strand. Buffer solution , providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations , magnesium or manganese ions; generally Mg 2+ is used

8. Principles and Procedures of PCR Cont’ PCR consists of a series of 30 to 40 but usually 35 repeated temperature changes called cycles. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis. Most commonly PCR is carried out with cycles that have three temperature steps. The cycling is often preceded by a single temperature step (called hold ) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. i.e. enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, Melting temperature of the primers.

9. Principles and Procedures of PCR Cont’ NB: Thaw all the reagents at room temperature and put them on an ice bucket Make a master mix depending on the number of samples for the PCR reaction ( MgCL,dNTPs, Buffer, F primer R primer, DNA template. Consitution of the mastermix is done on ice to minimize the commencement of any reactions. Taq polymerase is added last as it is sensitive to the buffer and reactions begin immediately it is added.

10. Principles and Procedures of PCR Cont’ Initialization step: This step consists of heating the reaction to a temperature of 94-96°C which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA. Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

11. Principles and Procedures of PCR Cont’ Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, and commonly a temperature of 72°C is used. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. Extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified . Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction products.

12. An illustration of the polymerase chain reaction (PCR).  Step A: Solution is heated to 95°C to denature ("unzip") the two strands of the target DNA (A).  Step B: Solution is cooled to ~55°C to allow the primers to anneal (bind) to the ends of the DNA strands (B). Step C:   Solution is reheated to ~75°C to allow TAQ polymerase to synthesize complementary copies of each strand. 

13. A strip of eight PCR tubes, each containing a 100μl reaction. Thermal cycler for PCR

14. Example of PCR results (bands)

15. Que??? How do you check whether the PCR generated the anticipated DNA fragments or amplicons?

16. PCR Optimization PCR can fail for various reasons due to its sensitivity to contamination causing amplification of spurious DNA products Contamination with extraneous DNA spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, sterilize, and thoroughly cleaning the work surface between reaction setups. Right primer sequence

17. APPLICATIONS OF PCR PCR is a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include: DNA cloning for sequencing DNA-based phylogeny functional analysis of genes the diagnosis of hereditary diseases/Detection of variations and mutations in genes Identification of genetic fingerprints (used in forensic sciences and paternity testing) Detection and diagnosis of infectious diseases e.g. HIV (esp. in pediatrics) 1993 Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.

18. Time for a break !!!!!

19. Restriction Fragment Length Polymorphisms (RFLPs) Restriction enzymes are proteins (found in bacteria) that cut DNA in small fragments. They recognize a very specific sequence of DNA called restriction site e.g. AGCT. Wherever the particular sequence of a restriction site occurs in a DNA molec , the restriction enzyme will cleave at that specific location. RE are isolated from bacteria and named acccording to the organism, usually has 3 or 4 letters followed by a number. 1 st letter designates genus from which the enzyme was isolated, 2 nd two letters comes from the species e.g. Hind111 was isolated from Haemophilus influenza, while Ecor was isolated from Escherichia coli. The nos refer to the order that the enzyme was isolated from the organism i.e. Hind111 was the 3 rd enzyme to be isolated from H. influenza. These enzymes form the defense mechanism hence their function to cut DNA of foreign org that penetrates the bacteria cell. The method is based on digesting a mixture of PCR amplicons of a single gene using one or more restriction enzymes and detecting the size of each of the individual resulting terminal fragments

20. Examples of Restriction Enzymes Enzyme Source Recognition Sequence Eco RI Escherichia coli 5'GAATTC 3'CTTAAG   Eco RII Escherichia coli 5'CCWGG 3'GGWCC   Bam HI Bacillus amyloliquefaciens 5'GGATCC 3'CCTAGG Hin dIII Haemophilus influenza 5'AAGCTT 3'TTCGAA

21. Restriction Fragment Length Polymorphisms (RFLPs) RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest. Restriction enzymes are DNA-cutting enzymes found in bacteria. They cut DNA at precise points producing: a collection of DNA fragments of precisely defined length. These fragments can be separated by electrophoresis, with the smaller fragments migrating further than the larger fragments. One or more of the fragments can be visualized with a "probe" — a molecule of single-stranded DNA that is complementary to a run of nucleotides in one or more of the restriction fragments and is radioactive (or fluorescent).

22. Sickle cell disease is a genetic disorder in which both genes in the patient encode the amino acid valine (Val) in the sixth position of the beta chain (beta S ) of the hemoglobin molecule. "Normal" beta chains (beta A ) have glutamic acid at this position. The only difference between the two genes is the substitution of a T for an A in the middle position of codon 6 as shown below: Analysis of RFLP variation is an important tool in genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting, and paternity testing.

23. Random Amplified Polymorphic DNA (RAPD) Markers Developed for the identification of genetic polymorphism Based on the polymerase chain reaction (PCR) RAPD markers are amplification products of anonymous DNA sequences using single, short and arbitrary oligonucleotide primers, and thus do not require prior knowledge of a DNA sequence. Low expense, efficiency in developing large numbers of DNA markers in a short time and requirement for less sophisticated equipment reproducibility of the RAPD profile is still the centre of debate!. The discovery that PCR with random primers can be used to amplify a set of randomly distributed loci in any genome has facilitated the development of genetic markers for a variety of purposes

24. Random Amplified Polymorphic DNA (RAPD) Markers Cont’ The standard RAPD technology utilises short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are generally separated on agarose gels and stained with ethidium bromide. Differ from the standard PCR condition in that only a single oligonucleotide of random sequence is employed and no prior knowledge of the genome subjected to analysis is required.

25. Random Amplified Polymorphic DNA (RAPD) Markers Cont’ The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence. RAPD markers are therefore dominant applications of RAPD Analysis

26. Random Amplified Polymorphic DNA (RAPD) Markers Cont’ Limitations of RAPD Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). PCR is an enzymatic reaction, therefore the quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible. Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

27. Assignment 1) Describe the following: a) Nested PCR b) Real time PCR 2) What is the role of each of the following components of PCR ? a)template b)primers c)Taq polymerase dNTPs 3) Draw the steps of PCR , and show how this allows you to amplify a specific region of DNA

28. Thank you, wish you well in your studies and God Bless