DNA FingerPrinting & DNase I Footprinting.pptx

DNA Fingerprinting process, DNase I Footprinting

1. DNA FINGERPRINTING & DNASE I FOOTPRINTING GENOMICS & PROTEONOMICS Jitika Gupta 24/IBT/02

2. BASIC OF DNA STRUCTURE Double Helix Structure : DNA is made of two strands forming a helical shape . Nucleotides : Adenine (A), Thymine (T), Cytosine (C), Guanine (G ) . Unique Sequences : The order of these nucleotides is unique to each person .

3. DNA FingerPrinting 01 Method used to identify individuals based on their unique DNA patterns. Also known as DNA profiling or genetic fingerprinting. Works with entire genomes or specific regions of an organism’s DNA. PCR , STRs , RFLP used for analyzing genome Forensic science, paternity testing, and genetic relationship studies.

4. 1.DNA Extraction 1.DNA Extraction Overview : The first step is to isolate DNA from biological samples. Common sources include blood, saliva, hair follicles, skin cells, or bodily fluids. Method : Basic components of extraction buffer include Tris , NaCl , SDS, EDTA, and water . EDTA inhibits nucleases from denaturing the DNA.  Tris make it permeable . Proteinase K are added hydrolyze histone proteins breaks peptide bonds of protein structure & aids to lysis of cell membrane . Detergents like SDS lyse the cell membranes, separate histone proteins from DNA, Denature histone proteins, and disrupt secondary and tertiary structures of proteins. The phenol-chloroform- isoamyl alcohol in the ratio 25:24:1 is mixed with the extract and centrifugated to separate the DNA molecules. The lower dense phase is the phenol-containing denatured proteins that are subsequently hydrolyzed with proteinase K. The intermediate separating layer contains the lipid dissolved in the chloroform, and the DNA remains in the top aqueous phase.

5. 2.DNA Amplification (Polymerase Chain Reaction - PCR) 2.DNA Amplification (Polymerase Chain Reaction - PCR) Overview : Technique involves the enzymatic procedure, whereby specific desired regions of the DNA templates are replicated repeatedly corresponding to each cycle under the influence of heating and cooling of the sample supplemented with specifically designed primers and free nucleotides How it Works : PCR process involve the denaturation, annealing and extension. In denaturation step the double stranded DNA denature to form the single stranded form. Whereas, during annealing, the primers bind to the specific DNA sequence at specific temperature. Finally, during extension, DNA polymerase extends the primers by coping the complimentary target region using the deoxynucleotide triphosphate building blocks (dNTPs). The targeted regions of the DNA molecule mostly contain tandem repeats. For human identification purpose, two types of tandem repeats are used, i.e., minisatellites commonly known as variable number tandem repeats (VNTR) and microsatellites commonly known as short tandem repeats (STR).

6. 3.DNA Fragmentation (Restriction Enzyme Digestion - RFLP) 3.DNA Fragmentation (Restriction Enzyme Digestion - RFLP) Overview : RFLP analyzes the length of the stands of DNA molecules with repetitive nucleotide bases by determining a specific pattern of repeat and then cut those into fragments with specific restriction enzymes .. How it Works : Restriction enzymes scan the DNA for particular sequences (usually 4-8 base pairs long) and cut the DNA at those sites. The result is a mixture of DNA fragments of various sizes. The specific locations and patterns of cuts depend on individual variations in the DNA sequence. Key Points : The size and number of DNA fragments differ . These fragments are then separated by electrophoresis which sorts fragments with respect to length. The segments are than radioactively tagged to produce visual pattern or “DNA fingerprint” on X-ray film

7. Short Tandem Repeat (STR) Analysis Short Tandem Repeat (STR) Analysis What Are STRs? Definition : STRs are short sequences of 2-6 base pairs that repeat in tandem at specific locations (loci) in the genome. Example : The sequence "GATA" repeated multiple times (e.g., GATA-GATA-GATA) forms an STR. Highly Variable : The number of repeats at a particular locus is highly variable among individuals, making STRs highly useful for distinguishing between DNA samples . PCR Amplification STR loci are amplified using Polymerase Chain Reaction (PCR) . Primers flanking the STR regions ensure only the target sequences are amplified. The process results in millions of copies of the target STR regions, even from small or degraded DNA samples .

8. Key Advantages of STR Analysis Highly Polymorphic Gel Electrophoresis Fluorescent tags attached to the primers make the STRs visible when detected by a laser . The amplified DNA fragments are separated based on their size using gel electrophoresis . Visualization and Analysis The DNA fragments appear as peaks in an electropherogram . Each peak represents the number of STR repeats at each locus . Key Advantages of STR Analysis Highly Polymorphic : STRs provide a high level of individual specificity. Efficient : The process is fast, reliable, and works with small or degraded DNA samples. Widely Used : STR analysis is the gold standard in forensic labs and legal cases worldwide. Comparison : RFLP (Restriction Fragment Length Polymorphism) : DNA is fragmented using restriction enzymes, and the resulting fragments are analyzed. This technique is slower and requires larger samples. STR Analysis : Uses PCR to amplify specific STR regions, making it faster, more sensitive, and able to work with degraded DNA or smaller samples.

9. 4.Separation by Gel Electrophoresis 4.Separation by Gel Electrophoresis Overview : Once DNA is fragmented or amplified, it needs to be separated by size. This is done using a technique called gel electrophoresis. How it Works : DNA samples are loaded into wells in a gel made of agarose or polyacrylamide. An electric current is passed through the gel, causing the negatively charged DNA fragments to move towards the positive end. Smaller DNA fragments move faster through the gel than larger ones, separating the fragments by size. The separated fragments form distinct bands, representing different DNA lengths. Key Points : The distance traveled by the DNA fragments is directly related to their size. The resulting pattern of bands forms a DNA profile unique to the individual.

10. 5.Visualization (Staining or Fluorescence) 5.Visualization (Staining or Fluorescence) Overview : After electrophoresis, the DNA fragments must be made visible to analyze the pattern. How it Works : The gel is stained with a dye (e.g., ethidium bromide or SYBR Green) that binds to DNA and fluoresces under UV light. Alternatively, if fluorescent tags were used in PCR, the DNA bands can be directly visualized without staining. The pattern of bands, which is the "DNA fingerprint," is then photographed or scanned for analysis. Key Points : Each individual has a unique pattern of DNA bands . The DNA profile can now be compared to other samples or reference databases.

11. Protozoa multiply very quickly 6.Analysis and Comparison Overview : The final step is to analyze the DNA fingerprint by comparing it to other samples or profiles. How it Works : In forensic cases, the DNA profile from a crime scene is compared to the DNA of suspects, victims, or a database. In paternity tests, the child’s DNA is compared to the potential father’s DNA to determine biological relationships. For wildlife studies, genetic diversity can be assessed by comparing profiles across different individuals in a population. Key Points : DNA profiles are compared based on the number and position of STR bands or RFLP patterns. A match between profiles can identify or exclude individuals with high accuracy.

12. Protozoa multiply very quickly Limitations of DNA Fingerprinting: Contamination of Samples : Can lead to inaccurate results. Identical Twins : DNA profiles are identical, making it hard to differentiate. DNA Databases: CODIS (Combined DNA Index System): National database in the U.S. for storing DNA profiles. Helps solve crimes by comparing DNA from crime scenes to known offenders. Other National and International Databases : Used for tracking criminals and missing persons . Functions : Personal Identification Diagnosis of inherited disease Breeding programs AIDS detection Development of cures for inherited disorders

13. Dnase I FootPrinting 02

14. Introduction Deoxyribonuclease I (DNase I) footprinting In the technique, a suitable uniquely end-labeled DNA fragment is allowed to inter act with a given DNA-binding protein and then the complex partially digested with DNase I. The bound protein protects the region of the DNA with which it interacts from attack by the DNase. Subsequent molecular-weight analysis of the degraded DNA by electrophoresis and autoradiography identifies the region of protection as a gap. DNase I footprinting can not only be used to study the DNA interactions of purified proteins but also as an assay to identify proteins of interest within nuclear extract. DNA fragments can be conveniently 5' labeled with T4 polynucleotide kinase and 3' labeled using the Klenow ( DNA Polymerase I, Commonly used with labeled dNTPs) or the T4 DNA polymerases or terminal transferase.

15. Protozoa multiply very quickly Experimental Procedure Binds in the minor groove of the DNA and cuts the phosphodiester backbone of both strands independently. Amplification of genomic DNA of interest by using PCR Labeling at the end of each strand of amplified DNA Protein of interest is added DNA is cleaved by DNase I nuclease Fragment run on Gel Electrophoresis & Autoradiography Applications Uses of DNase I Footprinting Identifying transcription factor binding sites Studying gene regulation Mapping protein-DNA interactions Identify functional gene in human genome

16. Protozoa multiply very quickly Result Selective Cleavage by DNase I : Certain regions, typically those that are more exposed in the DNA structure, are more susceptible to cleavage, while others, particularly where proteins are bound or the structure is tighter, remain less affected or uncut. This results in varying cleavage patterns, depending on the sequence and any bound proteins . Electrophoresis : When you compare the digestion products of naked DNA to protein-bound DNA, you typically see a "footprint" where the protein protected the DNA from being cleaved. This "footprint" is seen as missing bands or gaps in the ladder pattern of digestion products . Uneven Ladder : The ladder that appears after electrophoresis reflects the uneven nature of the cleavage. This pattern is not smooth but rather jagged, with some DNA fragments being more abundant (where cleavage occurred more easily) and others missing or reduced (where the DNA was protected by proteins).

17. References : DNase I Footprinting Benoît Leblanc and Tom Moss Techniques Involved in DNA Fingerprinting: Isolation, Quantification, PCR, Genotyping, and Analysis Braja  Kishore  Mohapatra Thankyou