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1. DNA Technology and Applications Dr. Nasser A. Elhawary Prof. of Medical Genetics
2. DNA CLONING Cloning of DNA segments:results in the production of a large no. of identical DNA molecules from a common ancestor. In vivocell-based DNA Cloning. In vitro PCR. Dolly was cloned by Ian Wilmut & Keith Campbell in 1996 Dolly (5 July 1996 – 14 February 2003) was a female sheep, and the first mammal to be cloned from an adult somatic cell, using the process of nuclear transfer
3. SmaI - - C C C G G G - - - - G G G C C C - - Blunt ends Background >>> Restriction Enzymes… EcoRI enzyme cuts the dsDNA segment into 2 sticky fragments) Restriction endonucleases (RE):Enzymes that recognize a specific base sequence in a DNA segment and cleave or nick the DNA at this site
4. Examples of Restriction endonucleases with their recognition sequence and cleavage sites
5. Background >>> Vectors… Vectors There are 5 main types of vectors commonly used which include plasmids, bacterio-phages, cosmids, bacterial (BACs) and yeast artificial chromosomes (YACs)
6. 1- In vivo cell based DNA cloning… Basic steps of in vivo DNA cloning: Generation of DNA fragments (using RE): Recombination of DNA fragments(DNA ligase). Vector:(A self-replicating DNA that is used to transfer foreign DNA segments between host cells). The incorporation of the target DNAinto a vector allows the production of large amounts of that DNA.
7. 1- In vivo cell based DNA cloning… 4. Transformation of the host organism: - The recombinant vector is introduced into specially modified bacterial host cells (Transformation process)(using potassium chloride). - If transformed cells multiply → large quantities of identical copies of the original single target DNA or clones. - The choice of vectors depends on a no. of factors, e.g. particular RE, the size of target DNA inserted.
8. 1- In vivo cell based DNA cloning… Screening for recombinant vectors. Selection of specific clones. What is DNA Library? It is the collection of recombin-ant of DNA molecules generated from a specific source, e.g. genomic or cDNA library. GeneBank:It is collection of clones that contains all the genetic information in an individual.
9. DNA Cloning…Summary
10. DNA Cloning… Summary
11. Polymerase Chain Reaction Kary Mullis, 1980; Nobel Prize 1993
12. 2- In vitro Polymerase Chain Reaction PCR is a cell-free DNA cloning. In 1985,PCR is a technique for amplifying a short segment of DNA (of order >106) in an in vitro reaction. It includes a three-step cycles. For 30 cycles, the dsDNA will give a PCR product of 230 (>109 copies). PCR-steps: 1- Denaturing 2- Annealing 3- Extension
13. 2- In vitro PCR…
14. 2- In vitro PCR…
15. RT-PCR RT-PCR (Reverse Transcription PCR): is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptaseto convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript.
16. Thermal Cycler Systems
17. Gel Electrophoresis Separate DNA fragments by their sizes (MW). Apply an electric field. DNA is -vely charged. Separate through gel matrix. Visualise DNA fragments under UV light.
18. Gel Electrophoresis of PCR products… Vertical Gel Unit To separate very close DNA fragments (1 bp) on Polyacrylamide gel (PAGE)
19. Solid Phase PCR Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers Transfer of PCR to ELISA plate
20. Quantitative DNA Concentration NANODROP Technology
21. PCR Applications DNA Amplifications Detection of disease mutations (using RE, ASO, SSCP, Denaturing HPLC, etc.) DNA sequencing (detect base-sequences) Gene expression studies Forensic medicine
22. RFLP in 1970 Restriction Fragment Length Polymorphism RFLP: is Variations in the length of DNA frag-ments generated by a Restriction Endonuclease (RE). Genetic variations almost never represent detectable differences that are functionally significant. Polymorphism is a form of a genetic variation due to the presence or absence of a cutting site.
23. Example of Genotyping (RFLPs) IVS10 mutation in PAH gene
24. DNA Fingerprinting… Jefffreys (1980) used a collection of probes that simultaneously detect variability in short, repeated DNA sequences at 8 to 15 loci scattered on different chromosomes. The resulting pattern of 25 to 60 bands is unique to each individual (except for identical twins). DNA Fingerprints describe this RFLP pattern of DNA fragments (forensics). Samples from blood, semen, hair, skin and tissues.
25. DNA Restriction enzyme digest - direction of migration + DNA Fingerprinting
26. Examples of DNA Hybridization & Probes Single Strand Conformational Polymorphism (SSCP)
27. Hybridization & Probes… Allele-Specific Oligonucleotide (ASO): Distinguishing between two DNA molecules differing by one base (N, M) by hybridization Hybridization will allow the ‘perfect match’ ASO to hybridize to its target. ASO with a single nucleotide with ‘mismatch’ will not hybridize.
28. ASO-Dot Blot…
29. Microarray Technology… It indicates that labeled DNA can be used to monitor the expression of DNA molecules attached to a solid support. It is defined as a high-throughput and versatile technology used for parallel gene expression analysis for thousands of genes of known and unknown function, to detect polymorphisms & mutations in gDNA.
30. Microarray Chip…
31. Patrick Brown at Stanford University 1990 Microarray Technology…
32. Microarray Chip… Glass cDNA microarray produced by using high-speed precision robot. This type of DNA microarray can bear between 10,000-20,000 spots (genes) on an area of 3.6 cm2.
33. Advanced DNA Technology Real-Time PCR: TaqMan™ and Light cycler™ use fluorescent tech to detect mutations by allelic PCR products. Ingradients: DNA, primers, probes, dNTPs, Buffers, Taq enzymes. Denaturing HPLC (DHPLC). DNA Sequencing. Next Generation Sequencing (NGS).
34. DHPLC technology… DHPLC-WAVE system.The columnSep separate the Heteroduplexes generating from a PCR product (a patient and wild type) at a critical temperature. It uses a polymer separating column, UV to detect PCR products.
35. DNA Sequencing
36. Determination of nucleotide sequence. Technique invented in 1970’s. F. Sanger - Termination method. Dideoxynucleotides. DNA Sequencing
37. Dideoxynucleotide 5’ BASE CH2 PPP O O 3’ No OH-group at 3’-end prevents strand extension
38. In 1970 DNA Sequencing… It is the ‘Gold standard’ to screen mutation using the dideoxy chain termination method. Denatured PCR (ssDNA) forms a cDNA using primers in a PCReaction. ddNTP’s labeled with fluorescent dyes (4 colors) are used to terminate the PCR reaction. The reaction products are separated on PAGE or capillary electrophoresis. Identify from 400-1000 basepairs.
39. Sanger sequencing reaction Single stranded DNA is amplified in the presence of fluorescently labeled ddNTPs that serve to terminate the reaction and label all the fragments of DNA produced. The fragments of DNA are then separated via polyacrylamide gel electrophoresis and the sequence read using a laser beam.
40. DNA Sequencing…
41. Shotgun Sequencing It is a top from 1995-2005 DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method to obtain reads. Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing. Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence. Shotgun sequencing was one of the precursor technologies that was responsible for enabling full genome sequencing.
42. Next-Generation Sequencing (NGS) Although ‘shotgun sequencing’ was the most advanced technique for sequencing genomes from about 1995–2005, NGS have surfaced. NGS produces shorter reads (anywhere from 25–500bp) but many hundreds of thousands or millions of reads in a relatively short time (on the order of a day). This results in high coverage, but the assembly process is much more computationally expensive. NGS technology are vastly superior to “shotgun sequencing” due to the high volume of data and the relatively short time it takes to sequence a whole genome. The major disadvantage of ‘NGS’ is that the accuracies are usually lower (although this is compensated for by the high coverage).