Bio lec 12

The source covers several topics related to biotechnology, focusing primarily on Genetic Engineering and Forensics. It also includes a brief mention of the Snow Trillium (Trillium nivale) as a random plant of the week. The Snow Trillium's name comes from the Latin word "nix" for snow, reflecting its blooming time in March when snow may still be present. It has a limited range in the midwestern US, is in the lily family, is found in areas with limestone-derived soils, and is a rare species in Ohio. The first specimen was discovered near Dublin along the Scioto River by John Riddell. DNA technology has a wide variety of uses.

The main focus is on the application of DNA technology in genetic engineering and forensics.

Genetic Engineering: Genetic engineering involves recombinant DNA technology, which combines genes from different sources.

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Recombinant DNA technology allows genes for specific protein products to be inserted into bacteria. The bacteria can then be grown to produce large quantities of the desired protein, such as insulin or proteins used as vaccines.

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It is also used to create genetically modified foods. Examples include "Bt" corn with built-in pesticide, "Golden Rice" with enhanced nutritional value, and tomatoes with a longer shelf life. Genetically engineered crops were introduced in 1996 and are now widely used; as of 2022, 95% of soy and 89% of corn crops in the US were genetically modified. GE crops are primarily used for insect resistance and herbicide tolerance. This technology is most commonly used to modify crop plants for traits like insect resistance (corn and cotton), increased vitamins (rice), and disease resistance (papaya and sweet potatoes).

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More unusual uses include glowing pet fish and allergen-free cats. The first transgenic dog, created in 2009, was a "Glowing Puppy" made by inserting a gene for fluorescence from a sea anemone into a dog egg cell.

How Recombinant DNA is Made:

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Bacterial plasmids are used as vectors (gene carriers). Plasmids are small, circular pieces of DNA that bacteria readily absorb.

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A desired gene is inserted into a plasmid.

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The modified plasmid is then isolated and exposed to bacteria, which absorb them.

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The bacteria with the modified plasmid will then express the desired gene.

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To insert genes into plasmids, restriction enzymes are used to cut the DNA. Restriction enzymes recognize short nucleotide sequences and cut the DNA at those specific sites.

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A staggered cut by a restriction enzyme results in "sticky ends". Sticky ends are single-stranded pieces of DNA at the ends of the double-stranded molecule that can readily connect with complementary sticky ends.

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Fragments can then be stuck back together using the DNA ligase enzyme.

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To generate sticky ends, the restriction enzyme's recognition sequence must be a palindrome, meaning it reads the same forwards and backward on both DNA strands (e.g., GAATTC/CTTAAG).

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Once the gene of interest is inserted into the plasmid, it can be absorbed by bacteria, which then express the gene. Bacteria can be grown readily to make many copies of the gene or to synthesize the protein encoded by the gene.

Gene Editing:

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The CRISPR-Cas9 system is a new technique for editing nucleotide sequences of genes in living cells.

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It can potentially be used to reveal the function of a gene or even to correct a mutation.

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Bacteria have a protein called Cas9 that can identify and cut out viral DNA. Cas9 can cut any DNA sequence that is complementary to a specific sequence of guiding RNA.

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A Cas9 protein combined with an RNA molecule complementary to a particular gene can be used to cut out a gene from a cell's chromosomes.

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After the cut, random DNA nucleotides are inserted by DNA repair enzymes, which "knocks out" the gene, making it non-functional. This process can help determine what the gene did in the cell.

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To edit a gene, researchers can add a segment of a normal gene along with the Cas9-guide RNA complex. After Cas9 cuts the target DNA, repair enzymes use the normal DNA segment as a template to repair the target DNA, potentially fixing a mutated gene.

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Researchers have used CRISPR-Cas9 to fix genetic defects in mice.

Forensics: Forensics is defined as the "scientific analysis of evidence for crime scene investigations and other legal proceedings".

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DNA Fingerprinting is one technique used in forensics to compare DNA samples.

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Every individual's genetic makeup is unique.

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A person's "DNA Fingerprint" is their unique collection of DNA fragments generated when restriction enzymes are used to cut up their DNA.

Generating a DNA Fingerprint:

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First, DNA evidence must be collected from various tissues like blood, semen, or skin.

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If the DNA sample is tiny, it often needs to be amplified (copied many times) before DNA Fingerprints can be generated.

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The Polymerase Chain Reaction (PCR) is used to copy DNA. Genomes are large, and typically only a small region is amplified.

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To amplify only a specific sequence, primers (short single-stranded DNA molecules) are used. Primers are selected to be complementary to the sequences at the ends of the target sequence, thus flanking it and marking the region to be copied. Only the DNA between the primers is amplified.

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After amplification, restriction enzymes are used to cut the DNA into pieces. Many different kinds of restriction enzymes exist, each recognizing and cutting at a specific sequence. Cuts can be straight across both strands or "staggered".

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Although humans are more than 99% genetically identical, the vast size of the human genome (about 3 billion base pairs) allows for enough variation (about 1 in 1000 difference, or 3 million differences between any two people) that differences can be detected.

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When DNA samples from two different people are digested with the same restriction enzyme(s), they will produce different numbers and sizes of DNA fragments.

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These different sets of DNA pieces with varying sizes are called Restriction Fragment Length Polymorphisms (RFLPs). RFLPs are differences (polymorphisms) in the size of the DNA fragments created by restriction enzymes.

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Gel Electrophoresis is then used to separate the DNA fragments by size. DNA is negatively charged due to its phosphate groups, so it moves through a gel towards an electrical current. Smaller pieces migrate faster than larger ones, resulting in a unique banding pattern for each individual.

Short Tandem Repeat (STR) Analysis:

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This is another technique commonly used in forensics.

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It analyzes repetitive sequences found between genes in the non-coding DNA (e.g., the sequence AGAT repeated multiple times).

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Close relatives are more likely to have more similar patterns of STRs.

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Standard STR analysis in forensics tests 13 different markers.

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The chance of two different, unrelated people being identical at all 13 markers is extremely low, estimated between 1 in a billion and 1 in several trillion.