DNA Fingerprinting Lecture - Structure & Extraction - University of Glasgow

DNA Technologies Cristina Gonzalez-Garcia [email protected]  Outline DNA Structure Extraction Manipulation (cutting) Analysis Amplification Sequencing Editing Further reading...

DNA Technologies Cristina Gonzalez-Garcia [email protected]  Outline DNA Structure Extraction Manipulation (cutting) Analysis Amplification Sequencing Editing Further reading Berg, Tymoczo and Stryer “Biochemistry” 7th edition (2012) or 8th edition (2015)(W.H. Freeman and Co.) Alberts, B., et al (2008) “Molecular Biology of the Cell” 5th edition (Garland Press) Campbell and Reece (2008) “Biology” 8th Edition, The Benjamin/Cummings Publishing Co. The hall of fame of DNA discoveries 1948, Chemistry Arne Wilhelm Kaurin Tiselius 1962, Medicine Francis Harry Compton Crick, James Dewey Watson, Maurice Hugh Frederick Wilkins 1978, Medicine Werner Arber, Daniel Nathans, Hamilton O. Smith 1980, Chemistry Paul Berg, Walter Gilbert, Frederick Sanger 1993, Chemistry Kary B. Mullis, Michael Smith 2020, Chemistry E. Charpentier, J. A. Doudna DNA Application DNA profiling (fingerprinting) DNA profiling has become the gold standard in forensic science since the first case >30 years ago. Despite being dogged by sample processing delays because of forensic lab backlogs, the technique has gotten progressively faster and more sensitive: Today, investigators can retrieve DNA profiles from skin cells left behind when a criminal merely touches a surface. This improved sensitivity combined with new data analysis approaches has made it possible for investigators to identify and distinguish multiple individuals from the DNA in a mixed sample. And it’s made possible efforts that are under way to develop user-friendly instruments that can run and analyze samples in less than two hours.  This Was the Birth of DNA Profiling www.smithsonianchannel.co.uk/ DNA profiling for forensic applications Why is DNA a good unique fingerprint ? Structure and organization of DNA How can we obtain and “barcode” the DNA of an individual ? Extract, cut, amplify and quantify DNA How can we automate the process ? Lab-On-Chip devices How accurate is this methodology ? Ethical and forensic concerns (out of scope) DNA Structure and organization The hall of fame of DNA discoveries 1948, Chemistry Arne Wilhelm Kaurin Tiselius 1962, Medicine Francis Harry Compton Crick, James Dewey Watson, Maurice Hugh Frederick Wilkins 1978, Medicine Werner Arber, Daniel Nathans, Hamilton O. Smith 1980, Chemistry Paul Berg, Walter Gilbert, Frederick Sanger 1993, Chemistry Kary B. Mullis, Michael Smith 2020, Chemistry E. Charpentier, J. A. Doudna The Nobel Prize 1962 “For their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material." Rosalind Franklin At the centre of Rosalind Franklin’s tombstone in London’s Willesden Jewish Cemetery is the word “scientist”. This is followed by the inscription, “Her research and discoveries on viruses remain of lasting benefit to mankind.” https://www.nature.com/articles/d41586-020-02144-4 She died in 1958 of ovarian cancer, aged 37 Nobel 1962 Watson, Crick, Wilkins, Medicine Nobel 1982 Klug, Chemistry  Sugar backbone Structure of DNA Bases Photo 51  5’ end 3’ end DNA bases Pyrimidines (single ring) Cytosine Thymine Purines (double ring) Guanine Adenine Phosphate-deoxyribose backbone 3’ end 5’ end DNA organization Each of us has enough DNA to reach from here to the sun and back, more than 300 times. How is all of that DNA packaged so tightly into a tiny nucleus? DNA organization Each of us has enough DNA to reach from here to the sun and back, more than 300 times. How is all of that DNA packaged so tightly into a tiny nucleus? Karyotype The human genome is organized in 23x2 chromosomes; 22 autosomes, while number 23 differs between females (XX) and males (XY). www.britannica.com DNA Structure and organization Why is DNA a good unique fingerprint ? Were to find uniqueness in the DNA ? The variability in coding sequences is kept as low as possible by evolutionary conserved mechanisms, to offer reliability and robustness to the human phenotype. 99% of the DNA is identical among individuals! Variability in the coding portion of DNA We can look at two main aspects to identify uniqueness 1. Mutations Rare but highly defined; not good for general fingerprinting, but might be hijacked for specific purposes 2. Degeneracy of the Amino Acid Code Inherent in the protein synthesis mechanism Degeneracy of the Amino Acid Code Transcription The genetic code has only ~20 amino acids Translation but 64 different codons are available Degeneracy of the Amino Acid Code Some changes in the DNA sequence do not impact the phenotype, so they are more likely to occur Degeneracy of the Amino Acid Code Degeneracy is typically on the third nucleotide that might have a role only when exchanging a purine with a pyrimidine, but not within them. Methionine is coded by the start codon. Why ? DOI: 10.1098/rsfs.2019.0038 Junk DNA ? Large portions of the genome that do not code for proteins: Between genes Within genes (introns) Searching for uniqueness in the nc portion Non-coding DNA Description Gene regulatory sequences Sequences that are involved in the process of transcription. Includes promoters, enhancers and silencers Non-coding RNA genes Codes for RNA molecules that are not translated into protein. Examples include genes for tRNA Introns Non-coding sequences within genes Are removed by RNA splicing prior to the formation of mRNA Telomeres Regions of repetitive DNA at the end of a chromosome Protects against chromosomal deterioration during replication Satellite DNA Tandemly repeating sequences of DNA (e.g. STRs) Structural component of heterochromatin and centromeres  Regulatory genes Searching for uniqueness in the nc portion Non-coding DNA Description Gene regulatory sequences Sequences that are involved in the process of transcription. Includes promoters, enhancers and silencers Non-coding RNA genes Codes for RNA molecules that are not translated into protein. Examples include genes for tRNA Introns Non-coding sequences within genes Are removed by RNA splicing prior to the formation of mRNA Telomeres Regions of repetitive DNA at the end of a chromosome Protects against chromosomal deterioration during replication Satellite DNA Tandemly repeating sequences of DNA (e.g. STRs) Structural component of heterochromatin and centromeres Searching for uniqueness in the nc portion Non-coding DNA Description Gene regulatory sequences Sequences that are involved in the process of transcription. Includes promoters, enhancers and silencers Non-coding RNA genes Codes for RNA molecules that are not translated into protein. Examples include genes for tRNA Introns Non-coding sequences within genes Are removed by RNA splicing prior to the formation of mRNA Telomeres Regions of repetitive DNA at the end of a chromosome Protects against chromosomal deterioration during replication Satellite DNA Tandemly repeating sequences of DNA (e.g. STRs) Structural component of heterochromatin and centromeres Searching for uniqueness in the nc portion Non-coding DNA Description Gene regulatory sequences Sequences that are involved in the process of transcription. Includes promoters, enhancers and silencers Non-coding RNA genes Codes for RNA molecules that are not translated into protein. Examples include genes for tRNA Introns Non-coding sequences within genes Are removed by RNA splicing prior to the formation of mRNA Telomeres Regions of repetitive DNA at the end of a chromosome Protects against chromosomal deterioration during replication Satellite DNA Tandemly repeating sequences of DNA (e.g. STRs) Structural component of heterochromatin and centromeres Telomere length shortens with age Searching for uniqueness in the nc portion Non-coding DNA Description Gene regulatory sequences Sequences that are involved in the process of transcription. Includes promoters, enhancers and silencers Non-coding RNA genes Codes for RNA molecules that are not translated into protein. Examples include genes for tRNA Introns Non-coding sequences within genes Are removed by RNA splicing prior to the formation of mRNA Telomeres Regions of repetitive DNA at the end of a chromosome Protects against chromosomal deterioration during replication Satellite DNA Tandemly repeating sequences of DNA (e.g. STRs) Structural component of heterochromatin and centromeres  Microsatellites (STRs) are a DNA profiling for forensic applications suitable target Why is DNA a good unique fingerprint ? Structure and organization of DNA How can we obtain and “barcode” the DNA of an individual ? Extract, cut, amplify and quantify DNA How can we automate the process ? Lab-On-Chip devices How accurate is this methodology ? Ethical and forensic concerns (out of scope)  Extract DNA profiling for forensic applications Cut Amplify Why is DNA a good unique fingerprint ? Quantify Structure and organization of DNA How can we obtain and “barcode” the DNA of an individual ? Extract, cut, amplify and quantify DNA How can we automate the process ? Lab-On-Chip devices How accurate is this methodology ? Ethical and forensic concerns (out of scope) How to analyse a DNA sample Extract the DNA from a (human) sample Blood, but it could be any biological material (urine, faeces, saliva, …) Cutting the DNA in meaningful slices Restriction enzymes DNA Amplification Polymerase chain reaction (PCR) Quantify DNA length Electrophoresis DNA Extraction Blood separation To isolate lymphocytes, whole blood is layered upon a density gradient medium of around 1.077 g/mL. Using a medium of this density will isolate all mononuclear cells (lymphocytes and monocytes). Following centrifugation, the neutrophils and RBCs will be pelleted at the bottom with a layer of mononuclear cells in the density medium above. RBCs: 5 106 cells/mm3 WBCs: 6 103 cells/mm3 DNA extraction Expose DNA (break the plasma and nuclear membranes), get rid of the proteins anchored to the DNA, separate the DNA from the debris  Cell membrane lysis DNA extraction flowchart Nuclear envelope lysis Protein digestion and debris removal Precipitation of DNA and resuspension  Cell membrane lysis 1. Membranes lysis Nuclear envelope lysis DOI 10.3390/mi8030083 Detergents (surfactants) Chemicals for DNA extraction Tris DNA is pH-sensitive, Tris buffer maintains the pH of the solution. Also, it interacts with the lipopolysaccharides of the cell membrane and makes them permeable, this will help in lysis of the cell membrane. EDTA EDTA is a chelating agent and can be used to block DNase activity. DNase is an enzyme which lyses the DNA. However, every enzyme required cofactor to work properly. SDS Sodium dodecyl sulphate is an anionic detergent which helps cell membrane and nuclear envelope to break open. NaCl DNA precipitation by reducing DNA solubility.  Cell membrane lysis 2. Protein digestion Nuclear envelope lysis Protein digestion and debris removal Proteinase K Proteinase K is an enzyme suitable to digest proteins in a detergent-rich environment. It is relatively unspecific and has a preference for aromatic and hydrophobic amino acid. Proteinase K dissolves DNA-bound proteins and DNAses which would fragment DNA How to analyse a DNA sample Extract the DNA from a (human) sample Blood, but it could be any biological material (urine, faeces, saliva, …) Cutting the DNA in meaningful slices Restriction enzymes DNA Amplification Polymerase chain reaction (PCR) Quantify DNA length Electrophoresis