Biotechniques (BMS 34010A) Fall 2023-2024 Lecture Notes PDF

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Basic Molecular Techniques  Learning outcomes  Define molecular biology methods and techniques.  Explain DNA cloning techniques.  Describe DNA and plasmid extraction.  Describe the...

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Basic Molecular Techniques  Learning outcomes  Define molecular biology methods and techniques.  Explain DNA cloning techniques.  Describe DNA and plasmid extraction.  Describe the CRISPR-Cas9 system.  Describe DNA synthesis and DNA microarray.  Explain the different types of DNA mutation.  Molecular biology methods  Molecular biology methods used to study the molecular basis of biological activity.  The essence of cell chemistry to isolate a particular cellular component and then analyze its chemical structure and activity.  Methods most commonly used to explore cells, their characteristics & processes: ▪ Nucleic acid methods. ▪ Protein methods. ▪ Immunostaining methods. Method is the approach or pathway (a process ). Technique is a man made strategy or tactic (practical aspects).  Molecular biology techniques  DNA cloning:  Gel electrophoresis:  Molecular hybridization: ▪ Cut & paste DNA ▪ Various types ▪ Southern blot ▪ DNA or RNA isolation ▪ Northern blot ▪ Ligation  Reading and writing DNA: ▪ Western blot ▪ Bacterial transformation or transfection ▪ Chromosome integration ▪ DNA sequencing ▪ Expression cloning ▪ DNA synthesis  Cell culture: ▪ Cellular screening  Polymerase Chain Reaction (PCR):  Rewriting DNA: mutations ▪ DNA polymerase DNA dependent ▪ Random mutagenesis  Arrays: ▪ PCR dynamics ▪ Point mutation ▪ DNA array ▪ PCR types ▪ Chromosome mutation ▪ Protein array ▪ CRISPR/Cas9  DNA cloning overview The inserted DNA is reproduced along with the vector Cut and paste DNA  Join two molecules togeth er: ▪ insert : usually smaller ▪ vector : has origin of replication  Two types of vectors are most Circular double stranded plasmid DNA Recombinant vector commonly used: (Cloning vector) ▪ E. coli plasmid vectors. ▪ bacteriophage λ vectors.  Cut and paste DNA: plasmids  Plasmids are circular, self-replicating, double-stranded DNA (dsDNA) m olecules th at are separate from a cell’s chromosomal DNA.  Plasmids have b een engineere d to optimize their use as vectors (reduced length ≈3kb; contain only the essential nucleotide sequences): ▪ restriction sites ▪ marker genes for selection and/or screening ▪ origin of replication  Plasmid vectors replicate along with their host cells (low, medium or high copy number). Cut and paste DNA: plasmids  A DNA fragment of a few base pairs up to ≈20 kb can be inserted into a plasmid vector.  Like the host-cell chromosomal DNA, pDNA is duplicated before every cell division. Cut and paste DNA: euk expression vectors  Eukaryote: viruses ▪ restriction sites ▪ virus genes ▪ terminal repeats  Cut and paste DNA: restriction  Restriction sites are specific 4- to 8-bp sequences that are recognized by restriction endonucleases (restrictases).  Many restriction sites are short inverted repeat sequences.  Restriction enzyme type 2 cut DNA: ▪ highly specific (type II endonucleases) ▪ leaves blunt or sticky ends Cut and paste DNA: restriction Cut and paste DNA: restriction  Cut and paste DNA: restriction ▪ DNA ligase catalyzes formation of 3’ → 5′ covalent bonds between the short fragments. ▪ Restriction fragments are covalently ligated together: ▪ Restriction fragments with complementary “sticky ends” are ligated easily. ▪ Blunt-end ligation requires a higher DNA concentration than ligation of sticky ends. DNA transfer into cells  Chromosome integration  Integration of the target genes into the host chromosome. ▪ preferable strategy to overcome the drawbacks of plasmid-based overexpression. ▪ plasmid-free stable mutants. ▪ possible in bacteria but usually necessary in eukaryotes.  Integrase is the enzyme that splices the viral DNA into a cellular chromosome. Chromosome integration Transposon- mediated gene transposition Homologous recombination a single Site-specific recombination is an exchange that crossover between a targeting gene occurs between pairs of defined sequences and a homologous DNA fragment on a (target sites). This process is mediated by a chromosome. The whole plasmid specific recombinase that can be expressed via sequence is integrated. a helper plasmid.  Cellular screening  Both vectors are derived from natural plasmids, but both have been genetically modified for convenient use as vectors. ▪ The plasmid pBR322 is simpler in structure. ▪ The pUC plasmid is a more advanced vector, whose structure allows direct visual selection of colonies containing vectors with donor DNA inserts. β-galactosidase is a protein encoded by the lacZ gene  DNA extraction  DNA extraction (isolation) is a method to purify DNA by using physical and/or chemical methods from a sample separating DNA from cell membranes, proteins, and other cellular components. ▪ The aim is to tak e only DNA f rom the whole cell extract.  The basic steps of DNA extraction: ▪ Lysis: the cell and the nucleus are broken open to release the DNA (mechanical disruption or chemical lysis with detergents, e.g. Proteinase K ). ▪ Precipitation: separation of DNA from the cellular debris (Na+ ions neutralize the negative charges on the DNA molecules & they are precipitated from aqueous solutions in ethanol or isopropanol). Silica membrane ▪ Purification: rinsed with alcohol to remove any remaining unwanted material. ▪ Elution: with either the elution buffer or with water.  Extraction of plasmids  Purification of plasmid DNA from bacterial DNA is based on the differential denaturation of chromosomal and plasmid DNA using alkaline lysis in order to separate the two. ▪ Neutralization with potassium acetate allows only the covalently closed plasmid DNA to reanneal and to stay solubilized. ▪ Chromosomal DNA and proteins removed by centrifugation. Dirty minipreps DNA cloning overview Crispr/Cas9  CRISPR-Cas9 was adapted from a naturally occurring genome editing system in bacteria. ▪ A Cas enzyme for cutting the target sequence. ▪ A single guide RNA (sgRNA), which binds to the target sequence of 20bp (PAM sequence).  CRISPR/Cas9 creates specific double-strand breaks at the target locus. These corrections result in two types of genome modifications: DNA synthesis  Commercial synthesis  Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.  Gene synthesis - $0.09/bp  DNA array Expression profiles  A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time. ▪ Probe is DNA molecules of variable length on a solid support (oligo chip). ▪ Sample is labeled DNA or RNA that will bind to the probes.  The principle behind microarrays is that complementary sequences will bind to each other.  DNA array  DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene. Clustering of expression profiles defines breast cancer cell line subtypes. https://doi.org/10.1371/journal.pone.0006146  Mutations and causes Mutations are alterations in DNA sequences that result in changes in the structure of a gene.  Spontaneous: ▪ At low frequency owing to 1) the chemical instability of purine and pyrimidine bases and 2) to errors during DNA replication.  Induced: ▪ Exposure of an organism to certain environmental factors may increase the frequency of spontaneous mutations. ✓ Chemical mutagens induce point mutations ✓ Ionizing radiation gives rise to large chromosomal abnormalities.  Main types of mutations  In biological systems that are capable of reproduction, we must first focus on whether mutations are heritable (offspring, and further descendants).  By the cell type where mutations occur, they are classified as: ▪ Germline mutations occur in gametes. ▪ Somatic mutations occur in other body cells.  By the size of the involved region, mutations can be classified as: ▪ Point mutations ▪ Chromosomal mutations nuclear DNA mutations ▪ Copy number variation (CNV) vs mtDNA mutations  Point mutations  Substitution: One base is incorrectly added during r eplication and replaces the pair in the corresponding position on the complementary strand.  Insertion: One or more extra nucleotides are inserted into replicating DNA, often resulting in a frameshift.  Deletion: One or more nucleotides is/are "skipped" during replication or otherwise excised, often resulting in a frameshift.  Point mutations  Missense (nonsynonymous): ▪ A single nucleotide resulting in a codon that codes for a different amino acid.  Nonsense: ▪ A single nucleotide resulting in a premature stop codon.  Synonymous: ▪ A single nucleotide that changes a codon to an amino acid with similar properties e.g. Lysine to Arginine.  Silent: ▪ A single nucleotide that does not alter amino acid sequences e.g. GCT, GCC, GCA and GCG all code for alanine.  Neutral: ▪ A single nucleotide that does not have any harmful or beneficial effect on the organism, it usually occurs at noncoding DNA regions.  Point mutations Mutations can also be categorized on the basis of the function:  The loss-of-function mutations cause a decrease or a loss of the gene product or the activity of the gene product.  The gain-of-function mutations cause an increase in the amount of gene product or its activity, and sometimes create a new property, leading to a toxic product responsible for a pathological effect.  Chromosomal mutations  Deletion: A region of a chromosome is lost, resulting in the absence of all the genes in that area.  Duplication: A region of a chromosome is repeated, resulting in an increase in dosage from the genes in that region.  Inversion: One region of a chromosome is flipped and reinserted.  Insertion: A region of a chromosome is cut from one chromosome and inserted into another. ▪ interchromosomal insertion - another non-homologous chromosome ▪ intrachromosomal insertion - a different region of the same chromosome  Translocation: A breakage in two chromosomes and each of the broken pieces reunites with another chromosome. Copy number variation (CNV)  Gene amplification: The number of tandem copies of a locus is increased.  Expanding trinucleotide repeat: The normal number of repeated trinucleotide sequences is expanded. Because CNVs change the structure of the genome, such mutations, together with inversions and translocations, are collectively classified as forms of genome structural variation.  Mutation hotspots  Genetic mutations are influenced by sequence context, structure, and genomic features.  Some areas of the genome tend to be more prone to mutations than others. These regions in a genome exhibit elevated rates of recombination relative to a neutral expectation.  These "hot spots" are often a result of the DNA sequence itself being more accessible to mutagens. Hot spots include areas of the genome with highly repetitive sequences, such as trinucleotide repeats.  Mitochondrial DNA mutations  In some cases, inherited changes in mitochondrial DNA can cause problems with growth, development, and function of the body's systems.  mtDNA mutations disrupt the mitochondria's ability to generate energy efficiently for cells. Human mitochondrial DNA (mtDNA) References  Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 7.1, DNA Cloning with Plasmid Vectors.  Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Site-Specific Recombination.  Griffiths AJF, Gelbart WM, Miller JH, et al. Modern Genetic Analysis. New York: W. H. Freeman; 1999. Cloning a Specific Gene.  Govindarajan R, Duraiyan J, Kaliyappan K, Palanisamy M. Microarray and its applications. J Pharm Bioallied Sci. 2012;4(Suppl 2):S310-S312. doi:10.4103/0975-7406.100283  Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 8.1, Mutations: Types and Causes.  Clancy, S. (2008) Genetic mutation. Nature Education 1(1):187  Mahdieh N, Rabbani B. An overview of mutation detection methods in genetic disorders. Iran J Pediatr. 2013;23(4):375-388.  Eichler, E. E. (2008) Copy Number Variation and Human Disease. Nature Education 1(3):1  Loewe, L. (2008) Genetic mutation. Nature Education 1(1):113