Analysis of DNA Lecture Notes - Republic of Iraq

Republic of Iraq Ministry of Higher Education Stage:S2 and Scientific Research Module: MGD Wasit Universty College of medicine :Lecture Title Analysis of DNA Lecturer Name: Dr –Dhamyaa K. Kadhim  Intended le...

Republic of Iraq Ministry of Higher Education Stage:S2 and Scientific Research Module: MGD Wasit Universty College of medicine :Lecture Title Analysis of DNA Lecturer Name: Dr –Dhamyaa K. Kadhim  Intended learning outcomes At the end of this lecture you should be able to: Describe in general terms a number of standard molecular processes, such as gene cloning, restriction analysis and DNA sequencing. (LO 9.1) Describe the theory behind DNA electrophoresis and how this technique can be used to provide information about DNA fragments. (LO 9.2) Explain PCR and appreciate its fundamental importance in genetic testing. (LO 9.3) Describe DNA hybridisation and appreciate its role of in genetic testing. (LO 9.4) Understand how PCR, restriction analysis and DNA hybridisation can be used in allele-specific tests. (LO 9.5)  Why do we need to know all this? ❖ Advances in molecular genetics approaches have enabled to the development of a wide range of approaches for identification and analysis of genes and specific alleles. ❖ To understand these approaches and their use in tracking genes through families it is important to comprehend some of the basic molecular techniques that are used in analysis of DNA such as: Restriction analysis Gene cloning, DNA sequencing DNA hybridisation (Southern blotting) Polymerase Chain Reaction (PCR)  Why do we need to know all this? ❖ Also it is important to comprehend analysis of DNA at chromosomal level such as: Karyotyping Fluorescent in situ hybridisation (FISH) 1. Analysis of DNA - at the gene level a) Restriction analysis / Gene cloning Restriction endonucleases (restriction enzymes) are bacterial enzymes that cut and recognise double-stranded DNA at a specific sequences (restriction site). Restriction enzymes were discovered, isolated and characterized in 1970s. Restriction sites are a sequence of DNA that can be recognised by a restriction endonuclease. The enzyme can then specifically cut the double stranded DNA molecule within (or outside) the recognition sequence. Restriction sites are often palindromic, and only a few nucleotides long (most often 4 or 6 bp, occasionally 8 or more bp). 1. Analysis of DNA - at the gene level a) Restriction analysis / Gene cloning Restriction enzyme recognises and cuts the sugar-phosphate backbone of the DNA strands to create single-stranded ends. The single-stranded tails are “sticky” and can form hydrogen bonds with complementary sticky tails on other DNA molecules cut with this enzyme. Close the gaps between the fragments was achieved by DNA ligase that enzyme seals the gaps in the phosphate backbone, creating a single recombinant DNA molecule 1. Analysis of DNA - at the gene level a) Restriction analysis / Gene cloning Restriction enzyme EcoRI recognises and cuts DNA molecules containing the following sequence: ↓ Restriction site: 5’GAATTC 3’ After cutting: 5’G AATTC 3’ 3’CTTAAG 5’ 3’CTTAA G 5’ ↑ 1. Analysis of DNA - at the gene level a) Restriction analysis / Gene cloning There are over 600 commercially available restriction enzymes: 1. Analysis of DNA - at the gene level a) Restriction analysis / Gene cloning Restriction enzyme are crucial tools in many molecular biological techniques such as gene cloning Gene cloning or recombinant DNA technology can be defined as introduction of a foreign DNA fragments into organism so that the population of organisms is created with same gene or genes present. 1. Analysis of DNA - at the gene level a) Restriction analysis / Gene cloning 1. Analysis of DNA - at the gene level b) Gel electrophoresis Gel electrophoresis: a technique which separates macromolecules (proteins/DNA/RNA) on the basis of their size or charge. Molecules are separated in a gel and migrate due to the presence of an electric charge placed across the gel. DNA molecules are negatively charged and they will move towards the positively electrode when placed in an electric field. DNA fragments can be separated according to size using this technique. Small fragments migrate quickly and larger fragments more slowly. After electrophoresis DNA can be visualised under UV light by staining with ethidium bromide (florescent dye), a chemical that binds to the DNA and fluoresces under UV light 1. Analysis of DNA - at the gene level b) Gel electrophoresis 1. Analysis of DNA - at the gene level c) Southern blotting / hybridisation Southern blotting: a technique where DNA separated by electrophoresis is transferred to a membrane filter and is detected by the binding of a labelled probe. It used to detect the variation in the large DNA fragment. Southern blotting involved the following steps: DNA molecule cutting with RE The resulting DNA fragments separated on the gel electrophoresis The separated DNA will denature into single strands, and then transferred from the gel to a solid positive membrane; this is refereed as blotting 1. Analysis of DNA - at the gene level c) Southern blotting / hybridisation Hybridisation: involved adding the probe: a single-stranded DNA molecule labelled with radioactive or fluorescent tags, this probe will be able to ‘find’ and hybridise with any specific complementary DNA sequences present on the membrane. 1. Analysis of DNA - at the gene level c) Southern blotting / hybridisation 1. Analysis of DNA - at the gene level d) Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR): a very powerful and sensitive technique whereby small fragments of DNA are amplified using a DNA (or RNA) template. The amount of template needed is minute; the amount of amplified DNA endproduct is enormous, enough for further experimental procedures, such as gene cloning, restriction analysis and DNA sequencing. PCR is the technique of choice for the diagnosis of many inherited diseases; it has also been used to detect the presence of tumour cells and the very early stages of infection by pathogenic microorganisms or viruses. 1. Analysis of DNA - at the gene level d) Polymerase Chain Reaction (PCR) PCR is sensitive which means that the template DNA amount can be minute and could even come from a very crude sample like a mouth swap (It allows the specific replication of a particular segment of DNA) PCR is needed two specific primers that must bind (hybridize) specifically to a known sequence on the template DNA. Primer: a short oligonucleotide (usually between 10-25 nt) that can be 3’-extended by DNA polymerase. Primers of specific sequence are used in molecular techniques like PCR and DNA sequencing. PCR is limited to amplifying segments of DNA up to 1000 bp. 1. Analysis of DNA - at the gene level: d) Polymerase Chain Reaction (PCR) Every PCR cycle consists of 3-step: 1. Denaturation: at high temperature(95°C) 2. Renaturation (annealing) the primer with DNA template: at lower temperature (55-60 °C) 3. DNA synthesis by DNA polymerase in the presence of deoxynucleotides: at medium temperature (72°C) This cycle will repeat for about 25-35 cycles There is an exponential increase in the amount of target DNA: 2n (n is the number of cycles). 1. Analysis of DNA - at the gene level: d) Polymerase Chain Reaction (PCR) 1. Analysis of DNA - at the molecular level DNA sequencing DNA sequencing is the ultimate analytical assay for DNA to determination of the individual nucleotides that are linked together to form the DNA molecule. Discovered in the early 1970s, and established fully in 1975 with the Sanger dideoxy chain termination method. DNA sequencing has now become a fully automated process, which was crucial for genomics: the study of entire genomes of organisms. It is important to detect the variation at DNA segment such as mutation 1. Analysis of DNA - at the molecular level DNA sequencing The sequencing reaction uses the enzyme DNA polymerase, one primer, the four deoxynucleotides (A, T, C, and G), and four modified nucleotides called dideoxynucleotides which do not have –OH on the 3' deoxypentose that needed to link nucleotides together, these modified nucleotides have an –H and labeled with a different fluorescent dyes. The primer binds to the single-stranded DNA and serves as the starting point for DNA polymerase to synthesize a new strand. 2. Analysis of DNA - at the molecular level DNA sequencing Automated Sequencing: Dideoxy chain termination Method '3 '5 1. Analysis of DNA - at the chromosome level a) Karyotyping A karyotype: is a picture of the full set of stained metaphase chromosomes of an individual organised according to chromosome number. It used to study the chromosomal abnormalities Numerical abnormalities structural abnormalities 1. Analysis of DNA - at the chromosome level a) Karyotyping Metaphase chromosomes Karyotyping 1. Analysis of DNA - at the chromosome level b) Fluorescent in situ hybridisation (FISH) This technique allows the investigation of specific DNA sequences on chromosomes inside the cell. Fluorescent probes for a specific gene (or several genes) can be used or probes for specific DNA stretches on chromosomes, like telomeres or centromeres. Chromosome 19 specific probe metaphase interphase 1. Analysis of DNA - at the chromosome level b) Fluorescent in situ hybridisation (FISH) Chromosome painting: is often used for chromosome investigation, whereby each chromosome is visualised using a different coloured fluorescent probe. THANK YOU