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Molecular geneticsMolecular genetics is a subfield of genetics that focuses on the structure and function of genes on a molecular level, including genetic variation, gene expression, and DNA replication and repair.This field aims to understand how genes are transmitted from one generation to the next and how they influence human behavior, health, and disease. Research in molecular genetics relies heavily on laboratory methods and technologies, such as DNA sequencing, PCR, and gene editing techniques.Molecular Genetics Methods1. Polymerase Chain Reaction (PCR)2. DNA sequencing (manual/automated)3. DNA Fingerprinting (DNA typing/profiling)4. Single nucleotide polymorphisms (SNPs)practical applicationsAmplify DNA for Cloning (PCR)✓ Amplify DNA for sequencing without cloning (PCR)✓ DNA sequencing reaction (PCR)✓ Mapping genes and regulatory sequences✓ Linkage analysis (identify genes for traits/diseases)✓ Diagnose disease✓ Pathogen screening✓ Sex determination✓ Forensic analysis✓ Paternity/maternity (relatedness)✓ Behavioral ecology studies (relatedness)✓ Molecular systematics and evolution (comparing homologous sequences in different organisms)✓ Population genetics (theoretical and applied)✓ Physiological genetics (studying basis of adaptation)✓ Livestock pedigrees (optimize breeding)✓ Wildlife management (stock identification/assessment)✓ Detection of Genetically Modified Food (GMOs)the Polymerase Chain Reaction (PCR)✓ Ability to generate identical high copy number DNAs made possible in the 1970s by recombinant DNA technology (i.e., cloning).✓ Cloning DNA is time consuming and expensive (>>$15/sample).✓ Probing libraries can be like hunting for a needle in a haystack.✓ PCR, “discovered” in 1983 by Kary Mullis, enables the amplification(or duplication) of millions of copies of any DNA sequence with known flanking sequences. ✓ Requires only simple, inexpensive ingredients and a couple hours.DNA templatePrimers (anneal to flanking sequences)DNA polymerasedNTPsMg2+Buffer✓ Can be performed by hand or in a machine called a thermal cycler.✓ 1993: Nobel Prize for ChemistryHow PCR works:1. Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence.2. Next, denature the DNA to single strands at 94˚C.3. Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA.4. Extend primers at 70-75˚C using a heat-resistant DNA polymerase such as Taq polymerase derived from Thermus aquaticus.5. Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220)to 35 trillion copies (245) of the target DNA.6. Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely.  7. Cool to 4˚C and store or use amplified PCR product for analysis.Example thermal cycler protocol used in lab:Step 17 min at 94˚C Initial DenatureStep 245 cycles of:20 sec at 94˚C Denature20 sec at 52˚C Anneal1 min at 72˚C ExtensionStep 37 min at 72˚C Final ExtensionStep 4Infinite hold at 4˚C StorageDNA Sequencing✓ DNA sequencing = determining the nucleotide sequence of DNA.✓ Developed by Frederick Sanger in the 1970s.Manual Dideoxy DNA sequencing-How it works:1. DNA template is denatured to single strands.2. DNA primer (with 3’ end near sequence of interest) is annealed to the template DNA and extended with DNA polymerase.3. Four reactions are set up, each containing:1. DNA template2. Primer annealed to template DNA3. DNA polymerase4. dNTPS (dATP, dTTP, dCTP, and dGTP)4. Next, a different radio-labeled dideoxynucleotide (ddATP, ddTTP, ddCTP, or ddGTP) is added to each of the four reaction tubes at 1/100th the concentration of normal dNTPs.5. ddNTPs possess a 3’-H instead of 3’-OH, compete in the reaction with normal dNTPS, and produce no phosphodiester bond.6. Whenever the radio-labeled ddNTPs are incorporated in the chain, DNA synthesis terminates.7. Each of the four reaction mixtures produces a population of DNA molecules with DNA chains terminating at all possible positions. 8. Extension products in each of the four reaction mixutesalso end with a different radio-labeled ddNTP(depending on the base).9. Next, each reaction mixture is electrophoresed in a separate lane (4 lanes) at high voltage on a polyacrylamide gel. 10.Pattern of bands in each of the four lanes is visualized on X-ray film.11.Location of “bands” in each of the four lanes indicate the size of the fragment terminating with a respective radio-labeled ddNTP.12.DNA sequence is deduced from the pattern of bands in the 4 lanes.Automated Dye-Terminator DNA Sequencing:1. Dideoxy DNA sequencing was time consuming, radioactive, and throughput was low, typically ~300 bp per run.2. Automated DNA sequencing employs the same general procedure, but uses ddNTPs labeled with fluorescent dyes.3. Combine 4 dyes in one reaction tube and electrophores in one lane on a polyacrylamide gel or capillary containing polyacrylamide.4. UV laser detects dyes and reads the sequence.5. Sequence data is displayed as colored peaks (chromatograms) that correspond to the position of each nucleotide in the sequence.6. Throughput is high, up to 1,200 bp per reaction and 96 reactions every 3 hours with capillary sequencers.7. Most automated DNA sequencers can load robotically and operate around the clock for weeks with minimal labor.DNA Fingerprinting (DNA typing/profiling)✓ No two individuals produced by sexually reproducing organisms (except identical twins) have exactly the same genotype.Why?  ✓ Crossing-over of chromosomes in meiosis prophase I.✓ Random alignment of maternal/paternal chromosomes in meiosis metaphase I.✓ Mutation✓ DNA replication errors (same effect as mutation)