FINAL : ch10, analyzing the structure and function of genes

Gene technology involves the __ __ __

isolation and manipulation of DNA

Recombinant DNA technology is a cornerstone of gene technology, allowing for the combination of DNA from different sources. A prime example is the production of ___ by inserting the human insulin gene into Escherichia coli bacteria 4.

Humulin (human insulin)

restriction enzymes, origin, function, recognition sites and result

• Origin: Found in prokaryotes.

• Function: These enzymes act as molecular scissors, cutting DNA at specific recognition sites.

• Recognition Sites: Often palindromic sequences (reading the same forwards and backward on opposite strands).

• Result: Generates DNA fragments, often with "sticky ends" (short, single-stranded overhangs) that can anneal to complementary ends.

Gel Electrophoresis

• Purpose: Separates DNA fragments based on size.

• Mechanism: DNA fragments are loaded into a gel matrix and subjected to an electric current. Negatively charged DNA migrates towards the positive electrode. Smaller fragments move faster and farther than larger fragments.

• Application: Used in DNA fingerprinting, where DNA samples are cut into fragments, separated, and compared to DNA size markers for identification.

methods of DNA cloning

1. Using the Same Restriction Enzyme:

   • If a DNA fragment and a vector (like a plasmid) are cut with the same restriction enzyme, they will generate complimentary sticky ends.

   • The DNA fragment can then be inserted into the vector.

2. Using Different Restriction Enzymes:

   • If cut with different enzymes, the ends may not be complementary.

   • DNA polymerase and nucleotides can be used to fill in the gaps and create blunt ends, which can then be joined.

key components of DNA cloning

• DNA Ligase: An enzyme that joins DNA fragments together by forming phosphodiester bonds.

• Plasmids: Small, circular DNA molecules found in bacteria that can replicate independently of the bacterial chromosome. They serve as vectors for carrying foreign DNA fragments.

• Origin of Replication: A sequence on the plasmid necessary for its replication within the host cell.

DNA Libraries

collections of DNA fragments from an organism, stored in a host, typically bacteria.

Human Genomic Library

• Content: Contains the entire genome of an organism, including coding (exons) and non-coding regions (introns, repetitive sequences).

• Creation: Fragments of the entire genome are inserted into vectors and introduced into bacteria.

• Characteristics:

  • The same library can be created from any cell type of the organism.

  • Contains large amounts of non-coding DNA.

  • Useful for studying the overall genetic makeup of an organism.

Complementary DNA (cDNA) Library

• Content: Contains only the DNA sequences that were actively being transcribed into messenger RNA (mRNA) in specific cells. It represents the protein-coding DNA sequences.

• Creation:

  1. mRNA is isolated from cells.

  2. Reverse transcriptase synthesizes a DNA strand complementary to the mRNA (creating a DNA-RNA hybrid).

  3. DNA polymerase synthesizes a second DNA strand, forming double-stranded cDNA.

  4. This cDNA is then digested, added to plasmids, and transformed into bacteria for amplification.

• Characteristics:

  • Cell-specific: Different cell types produce different mRNAs, resulting in distinct cDNA libraries.

  • Indicates gene activity: Reveals which genes are active under specific conditions or in particular cell types.

Hybridization is a technique used to identify

a specific DNA or RNA sequence of interest within a complex mixture.

Hybridization relies on the

• complementary base pairing between two single strands of nucleic acids (DNA-DNA, DNA-RNA, or RNA-RNA).

process of hybridization

• Process:

  1. A probe is designed or selected. This probe is a labeled (e.g., fluorescently or radioactively) single strand of nucleic acid that is complementary to the target sequence.

  2. The DNA or RNA sample is denatured (separated into single strands, usually by heat).

  3. The labeled probe is added to the sample under conditions that allow hybridization (annealing of complementary strands).

  4. If the target sequence is present, the probe will bind to it.

  5. The location or presence of the target sequence can then be detected by visualizing the label on the probe.

Polymerase Chain Reaction (PCR) is a powerful technique used to

amplify (make millions or billions of copies of) a specific DNA segment in vitro.

explain the main 3 steps of PCR amplification

PCR involves repeated cycles of three main steps:

1. Denaturation: The double-stranded DNA sample is heated to high temperatures (e.g., $94-98^\circ C$) to separate the two strands.

2. Annealing: The sample is cooled (e.g., to $50-65^\circ C$) allowing primers (short, synthetic DNA sequences) to bind (hybridize) to their complementary sequences on the single-stranded DNA template. Primers define the specific region to be amplified.

3. Extension/Synthesis: The temperature is raised (e.g., to $72^\circ C$) for DNA polymerase (typically a thermophilic enzyme like Taq polymerase, isolated from thermophilic bacteria) to synthesize new DNA strands, starting from the primers. The enzyme uses the original DNA strand as a template and incorporates the four deoxynucleotide triphosphates (dNTPs: dATP, dCTP, dGTP, dTTP).

• Repetition: These three steps are repeated for 20-30 cycles. Each cycle theoretically doubles the amount of the target DNA sequence, leading to exponential amplification.

• Result: Billions of copies of the target DNA sequence are produced.

practical uses of PCR

Practical Uses:

• Tracking epidemics: Identifying and monitoring the spread of infectious diseases by amplifying pathogen DNA/RNA.

• Food testing: Detecting the presence of specific pathogens (e.g., bacteria, viruses) in food samples.

• Authenticity testing: Verifying the origin or species of food products.

• Detecting bioterrorist attacks: Identifying biological agents.

DNA fingerprinting uses variations in DNA sequences to identify individuals. A common method relies on

Short Tandem Repeats (STRs).

what are STRs?

• STRs: These are short DNA sequences (typically 4-40 base pairs in length) that are repeated multiple times in a row.

The Dideoxy (Sanger) sequencing method is used to determine the

precise nucleotide sequence of a DNA fragment.

Fluorescence In Situ Hybridization (FISH)

cytogenetic technique used to map the location of specific DNA sequences within cells and chromosomes. It can also be used to determine where a gene is being expressed.

applications of FISH

• Gene Mapping: Precisely locating genes on chromosomes.

• Detecting Chromosomal Abnormalities: Identifying deletions, duplications, translocations, and aneuploidies.

• Gene Expression: Detecting the presence of specific mRNA transcripts within cells, indicating gene activity.

Multicolor FISH: Uses multiple probes, each labeled with a different fluorescent dye, to simultaneously visualize different sequences or regions on the same chromosome or across multiple chromosomes.

Example: Detecting a chromosomal abnormality in Acute Lymphoblastic Leukemia (ALL). A tumor cell line might show only one red signal (for the CDKN2A gene) and one green signal (for the Centromere of chromosome 9) in an interphase nucleus, indicating a deletion of one copy of the CDKN2A gene on chromosome 9, whereas a normal cell would show two red and two green signals.

• Spontaneous Mutations

occur naturally

induced mutations

• Using mutagens (chemicals or radiation) to accelerate the mutation rate.

Model Organisms:

Commonly used in organisms with rapid generation times, such as bacteria, yeasts, fruit flies (Drosophila melanogaster), and nematodes (C. elegans).

RNA interference is

natural cellular mechanism that regulates gene expression, primarily by silencing specific genes. It can be harnessed experimentally to study gene function.

Transgenic organisms possess genetic material that has been altered or introduced from another source. This is often achieved through

gene knock-in or gene knockout strategies.

Gene Knock-in

• Process: Involves introducing a specific, often altered, gene into an organism's genome.

  1. A desired gene is introduced into Embryonic Stem (ES) cells.

  2. A few ES cells become transformed (contain the altered gene, often just one copy).

  3. These transformed ES cells are injected into an early mouse embryo.

  4. The resulting adult mouse may have some somatic cells (body cells) with the altered gene.

  5. If the alteration also occurs in germ-line cells (sperm or egg cells), the altered gene can be passed to offspring.

  6. Breeding these mice with normal mice produces progeny where some individuals have the altered gene in all cells (a "knock-in" mouse).

  7. Further breeding of knock-in mice can result in offspring with two copies of the altered gene.

Gene Knockout

• Process: Involves inactivating or completely deleting a specific gene from an organism's genome.

• Result: Allows researchers to study the function of the gene by observing the consequences of its absence.

• Example: Mice lacking the leptin gene. Leptin is a hormone that inhibits hunger. Mice without functional leptin become obese because they continuously feel hungry.

CRISPR-Cas9

is a powerful gene-editing tool derived from a bacterial defense system against foreign DNA (like viruses).

Creating genetically modified (transgenic) plants involves

introducing new genetic material into plant cells

process of transgenic plants

1. Infection with Agrobacterium: A portion of a plant leaf is incubated with Agrobacterium tumefaciens, a bacterium naturally capable of transferring a piece of its plasmid DNA (called the T-DNA) into plant cells.

2. Plasmid Modification: The Agrobacterium plasmid is engineered to contain a marker gene (e.g., for antibiotic resistance) and the altered gene of interest.

3. Transformation:Agrobacterium transfers the T-DNA (containing the desired gene) into the plant cells.

4. Callus Formation: The transformed plant cells are cultured on a special medium to form a callus – an undifferentiated mass of plant cells.

5. Regeneration: The callus is manipulated using plant hormones on different media to induce the development of whole plants. These regenerated plants carry the introduced gene.

Golden Rice Example

Golden Rice is a transgenic rice variety engineered to produce β-carotene(CCC1=CC(C)=C(C)C=C1)C(C)C[C@@H]2CC=C(C)C=C2] in the endosperm.

• Goal: To combat Vitamin A deficiency in populations where rice is a staple food.

• Enzymes involved: The engineered pathway involves enzymes that convert geranylgeranyl pyrophosphate into β-carotene.

• Issue: The initial version of Golden Rice produced β-carotene in the endosperm, but not necessarily in the form that could be readily converted to Vitamin A in the human body. Later versions aimed to improve this conversion.