molecular techniques in action MCQ

Applications of DNA Technology

• Foundational to medicine, agriculture, and environmental science.
• Gene Cloning makes multiple copies of a specific gene or DNA fragment, by inserting the gene into a vector, which is introduced into a host cell.
• Gene cloning is used to produce therapeutic proteins for medical treatments.
• Genetic Engineering manipulates an organism's genome to introduce, remove, or alter genetic material.
• Genetic engineering is often used to create genetically modified organisms (GMOs) like pest-resistant crops.
• Genetic engineering can also improve disease resistance or growth rates in animals.
• Gene Therapy introduces healthy genes into an individual’s cells to treat or prevent disease, often using a viral vector.
• Gene therapy can treat inherited diseases such as cystic fibrosis or sickle cell anemia.
• DNA fingerprinting, or profiling, is used in criminal investigations to match DNA from a crime scene to a suspect.
• It involves isolating and analyzing DNA to create a unique individual profile (except for identical twins).

Transgenic Technology

• Transgenic technology inserts foreign DNA into an organism’s genome to create transgenic organisms (TGOs).
• Transgenic plants gain beneficial traits through inserted genes from other organisms.
• Inserting the Bt gene from Bacillus thuringiensis into cotton plants makes them pest-resistant.
• Transgenic animals are genetically modified to include foreign genes.
• Transgenic animal genes are inserted to study diseases or produce valuable products.
• Some transgenic animals produce human proteins (like insulin) in their milk for medicinal use.
• Gene editing tools like CRISPR-Cas9 target and edit specific genes in living organisms, and have potential for correcting genetic mutations.

Nuclear Transfer Technology

• Nuclear transfer is a cloning technique where a somatic cell nucleus is transferred into an enucleated egg cell.
• Somatic Cell Nuclear Transfer (SCNT) removes a nucleus from an egg cell and replaces it with a nucleus from a somatic cell.
• SCNT creates an embryo genetically identical to the somatic cell donor.
• SCNT is used to create genetically identical animals for research or agricultural purposes.
• It can clone animals with desirable traits or preserve endangered species.

Embryos and Embryonic Stem Cells

• Embryonic technology involves manipulating early-stage embryos.
• Embryonic cloning splits a blastocyst to create multiple genetically identical organisms.
• Embryonic stem cells from early-stage embryos can differentiate into any cell type (pluripotent).
• Embryonic stem cells are a resource for regenerative medicine to replace damaged tissues or organs.

Transgenic Animals

• Transgenic animals are genetically modified to include foreign DNA to study diseases or produce useful substances.
• Transgenic animals are used as research models to study human diseases such as Alzheimer's or cystic fibrosis.
• Some transgenic animals produce human proteins or other substances.
• Goats can be genetically modified to produce human antithrombin (a protein used to treat blood clotting disorders) in their milk.
• Transgenic animals can be created with resistance to diseases.
• Inserting genes can prevent viral infections in livestock.
• Xenotransplantation transplants organs from one species to another (e.g., from pigs to humans).
• Transgenic pigs are used to make their organs more suitable for human transplant and reduce organ rejection risk.
• Cloning can replicate high-performing animals, preserving and passing on desirable traits.
• Cloning endangered species and creating transgenic animals can preserve biodiversity.

Gene Therapy and Retrovirus-based Gene Therapy

• Gene therapy inserts a functional copy of a gene into a patient’s cells to treat or prevent disease.
• Retroviruses can insert their RNA into the DNA of the host cell.
• Retroviruses are commonly used as vectors for gene therapy.
• In gene therapy, a retrovirus can carry a healthy gene into a patient’s cells.
• The retrovirus integrates its RNA into the patient’s genome.
• Viruses like retroviruses, adenoviruses, and lentiviruses are engineered to deliver therapeutic genes without causing disease.

Vectors and Plasmids

• Plasmids are small, circular DNA pieces in bacteria, separate from chromosomal DNA.
• Plasmids can be manipulated to introduce foreign DNA into cells.
• Vectors like plasmids insert new genes into bacteria, yeast, or mammalian cells.
• Plasmids can carry genes for antibiotic resistance, which allow researchers to select for cells that have successfully taken up the plasmid.
• Restriction Enzymes such as EcoRI, PstI, and SalI cut DNA at specific sequences, making it easier to insert foreign genes.
• Genes for antibiotic resistance are often included in plasmids.
• Only bacteria that have successfully taken up the plasmid will survive when grown in media containing the antibiotic.
• Multiple Cloning Sites (MCS) are regions in plasmids with multiple restriction enzyme recognition sites.
• MCS makes it easier to insert foreign DNA into the plasmid.
• Plasmids are extra circular DNA in bacteria that bacteria use to exchange helpful genes, such as those for antibiotic resistance.
• Scientists use plasmids as vectors to introduce new genes into bacteria or other cells.
• A vector is a delivery truck that transports genetic material into cells.
• Restriction enzymes cut DNA at specific points and function like scissors
• Selection markers are a security check; only bacteria with the plasmid survive, (e.g., antibiotic resistance).
• MCS act like a power strip, allowing easy insertion of new genes

Conformation of DNA

• DNA can exist in various forms influencing its function and stability, depending on structure and supercoiling.
• Nicked Circular DNA (Single-Strand Cut): A plasmid with a single-strand break in one of its two strands.
• Nicked circular DNA is slightly relaxed, and easier for cells to take up, which makes them useful in cloning experiments.
• Linear DNA (Double-Strand Cut): DNA that has been cut on both strands, making it no longer circular.
• Linear DNA is essential for inserting genes into other DNA molecules or genomes.
• Linear DNA is more stable for gel electrophoresis and for integrating DNA into a host genome in genetic engineering.
• Supercoiled DNA is naturally twisted and tightly wound, and helps DNA fit inside the nucleus of cells.
• Supercoiling is crucial for the stability and function of the DNA.

Viral RNA and Retrovirus

• Retroviruses are used in gene therapy due to their ability to integrate into the host genome.
• Retroviruses can carry therapeutic genes into human cells.
• They infect cells by attaching to their surface and releasing their RNA into the cell, which then gets converted into DNA and integrated into the host genome.
• Retroviruses are engineered to carry therapeutic genes and deliver them to specific tissues.
• This process is explored to treat genetic disorders like cystic fibrosis and sickle cell anemia.

Genomic DNA

• Genomic DNA is the complete set of DNA in an organism, containing all of its genes and non-coding sequences.
• Genomic DNA is isolated from cells and cut into smaller fragments to make it easier to analyze and manipulate.
• Genomic DNA is cut into smaller pieces using restriction enzymes, which recognize specific DNA sequences.
• Small fragments of genomic DNA are inserted into vectors for further study.
• The fragments can be cloned, meaning they are copied in a host cell, allowing researchers to analyze individual genes or DNA sequences in greater detail.

mRNA (Messenger RNA)

• mRNA plays a crucial role in protein synthesis, carrying genetic information from DNA to ribosomes.
• mRNA Synthesis: mRNA is created in transcription, being a complementary copy of a gene or genes in DNA.
• cDNA (Complementary DNA) is synthesized from mRNA using reverse transcriptase.
• cDNA is used for further research or cloning, and does not contain introns, making it suitable for studying gene expression.

Recombinant DNA Technology

• Recombinant DNA technology combines DNA from different sources to create new genetic combinations.
• Scientists use vectors, such as plasmids, to insert foreign DNA into a host cell carrying genes or DNA fragments from another organism.
• Recombinant DNA can be introduced into a host cell and can be replicated to produce proteins or further studied.
• Example: Inserting a gene for human insulin into a plasmid and introducing it into E. coli bacteria.

Genomic Library

• Genomic library is a collection of DNA fragments from an organism’s genome, stored in vectors and introduced into a host organism.
• Construction of a Genomic Library occurs when an organism's genomic DNA is fragmented into small pieces, which are inserted into vectors and introduced into bacterial cells.
• Each bacterial colony in the library contains a different fragment of the organism's genome.
• cDNA Library is made by converting mRNA into cDNA, containing genes that are actively being expressed in a specific tissue or at a particular time.
• It is a way to study which genes are "on" and producing proteins in a particular cell or condition.

Polymerase Chain Reaction (PCR)

• PCR is a tool used to amplify a specific segment of DNA, widely used in molecular biology, forensics, and medical diagnostics.
• Process of PCR includes three main steps:
  • Denaturation: DNA is heated to around 90°C, causing the double-stranded DNA to separate into two single strands.
  • Annealing: The temperature is lowered, and the primers bind to the complementary sequences on the single-stranded DNA.
  • Extension: The DNA polymerase enzyme synthesizes new DNA strands from the primers, creating a copy of the target sequence.
• Each cycle of PCR doubles the amount of DNA, leading to the exponential amplification of the target region.

DNA Polymerase and Cloning

• DNA polymerase plays a critical role in DNA replication making new DNA strands.
• In Cloning, DNA polymerase is used in the PCR process to help create multiple copies of a DNA fragment.
• Cloning involves copying a gene or DNA fragment from one organism and inserting it into a new organism or cell.
• In Recombinant DNA, once the DNA is inserted into a host cell, it can be replicated to produce many copies of the gene of interest.

Denaturation and Binding of DNA

• Denaturation breaks the hydrogen bonds in the DNA double helix, unwinding into single strands.
• Heating to 90°C causes the DNA to separate into single strands in PCR.
• Binding of Primers occures when the temperature is lowered, allowing the primers to bind to the single-stranded DNA at the target region.
• Primers are nucleotide sequences act as a starting point for DNA polymerase to begin the new DNA strand.

Recombinant Plasmids

• Recombinant plasmid-a plasmid that contains foreign DNA inserted into it while serving as a vector for cloning new genetic material into cells.
• Plasmid Vectors are small, easy to manipulate, and can replicate independently in bacteria.
• Applications of Recombinant Plasmids can also be commonly be used in research, drug production, and genetic engineering.
• Recombinant plasmids can be introduced to produce insulin in E.coli bacteria.

Short Tandem Repeats (STRs)

• STRs are short sequences of base pairs found within the two coiled strands of DNA
• STRs are located within non-coding regions of the genome and are part of the double-helix structure
• Each person inherits STRs from both parents, making the number of repeats at each STR location unique
• Analysis
  • Collection: DNA is collected from a sample
  • PCR Amplification: Specific STR loci are amplified using PCR
  • Gel Electrophoresis: STRs are separated by size using gel electrophoresis
  • The gel matrix acts as a filter; smaller DNA fragments move faster than larger ones
• Comparison

PCR (Polymerase Chain Reaction)

• PCR amplifies specific segments of DNA, useful when only a small amount of DNA is available.
• PCR Steps:
  • Denaturation (94-98°C): Double-stranded DNA separates into single strands.
  • Annealing (50-65°C): Primers bind to complementary sequences.
  • Extension (72°C): DNA polymerase synthesizes a new DNA strand using Taq polymerase.
• Amplification: The process is repeated to increase the number of copies of the target DNA region.

DNA Fingerprinting

• Dna fingerprinting creates a unique genetic profile of an individual, and involves RFLP (Restriction Fragment Length Polymorphism) and STR analysis.
• RFLP Analysis Process:
  • Extraction
  • Cutting with Restriction Enzymes
• Understanding the Gel Electrophoresis to X-ray Exposure Process
  • Gel Electrophoresis: Separates DNA fragments by size using an electric current in a gel matrix, with smaller fragments moving faster than larger ones.
  • Southern Blotting: Transfers separated DNA fragments from the gel onto a nylon membrane.
  • DNA Probes and Labeling: Probes are small, single-stranded DNA sequences complementary to target sequences, and are labeled for detection.
  • Binding and Detection: Probes bind to target DNA fragments on the membrane, before exposing the membrane to X-ray film.
  • The radioactive or fluorescent tag on the probe allows to see where the probe bound, creating bands on film

Cloning: Cutting DNA

• DNA cloning isolates a specific gene or DNA fragment and inserts it into another organism’s DNA.
• Steps in Cloning:
  • Cutting DNA with Restriction Enzymes
    • Enzymes cut DNA at specific sequences, creating sticky ends or blunt ends
    • For example, EcoRI cuts GAATTC, leaving sticky ends that can easily be joined
  • Inserting the DNA into a Vector:
    • Inserting a DNA fragment into the vector using DNA ligase, an enzyme that seals the DNA fragment into the vector.
    • A vector carries foreign DNA into a host cell.
  • Transformation:
    • The recombinant vector is introduced into a bacterial cell.
    • The host cell contains foreign genetic material and has ability for replication.

Vectors

• Vectors are used to carry foreign DNA into a host organism.
• Plasmids: Small, circular DNA molecules that exist independently of chromosomal DNA in bacteria, and can carry foreign DNA.
• Plasmid Characteristics:
  • Origin of replication (ori): Allows the plasmid to replicate within a bacterial cell.
  • Selectable markers: Genes for antibiotic resistance are included to select for bacteria that have taken up the plasmid.
  • Multiple Cloning Sites (MCS): Regions with multiple restriction sites where foreign DNA can be inserted.
• Bacterial Artificial Chromosomes (BACs): Used to clone larger pieces of DNA, and Useful for creating genomic libraries.

Types of DNA Libraries

• DNA libraries are collections of DNA fragments stored in a host organism for further analysis.
  • Genomic Library contains all of the DNA from an organism's genome.
    • Each bacterium holds a different fragment of genetic material.
    • Allows Researchers to access every part of the genome for study
  • cDNA Library: Constructed from mRNA
    • Reverse Transcriptase is used to make cDNA from mRNA
    • Useful for studying gene expression since only contains expressed genes.

DNA Sequencing (Sanger Method)

• Sanger sequencing determines the exact sequence of nucleotides in a DNA molecule.
  • Steps:
    • Template DNA must be prepared
    • Primer- A short, single stranded DNA molecule binds to a start position on the DNA region.
    • Reaction Setup occurs to ensure it contains normal nucleotides (dATP, dTTP, dCTP, dGTP) and modified dideoxynucleotides (ddATP, ddTTP, ddCTP, ddGTP)
    • DNA Synthesis: DNA polymerase is created to incorporate and extend nucleotides leading to chain termination.
    • Electrophoresis: The resulting fragments are spearated by size.
    • Reading the Sequence takes place using gel electrophoresis.

Polymerase Chain Reaction (PCR)

• PCR is a laboratory technique used to amplify specific segments of DNA.
• Steps in PCR:
  • Denaturation (94-98°C): Double-stranded DNA template is heated, causing strands to separate.
  • Annealing (50-65°C): Temperature is lowered, allowing primers (forward and reverse) to bind to template DNA.
  • Extension (72°C): Taq polymerase (a heat-stable enzyme) synthesizes new DNA strands.
• Amplification Cycles: Repetition of denaturation, annealing, and extension amplify target DNA region.

DNA Cloning and Manipulation Techniques

• DNA cloning involves isolating and making copies of specific DNA sequences
  • Tools for Cloning:
    • Restriction Endonucleases (REs) are proteins that recognize and cut DNA at specific palindromic sequences.
    • DNA Ligase: Enzyme that joins DNA fragments, used after restriction enzymes cut the DNA.
• Vectors and Living Systems in Cloning:
  • Vectors deliver foreign DNA into a host cell. Common vectors include:
    • Plasmids: Small circular DNA molecules
    • Bacteriophages: Viruses
    • BACs (Bacterial Artificial Chromosomes).
  • Living Systems: Cloned DNA must be propagated in a living system, often in bacterial cells.

Cutting DNA with Restriction Endonucleases

• Restriction Enzyme: Cleaves DNA at specific recognition sequences, which are usually palindromic.
  • The product of cutting the DNA results in Sticky ends, or blunt ends.
• Fragments:
  • Use of DNA ligase is useful to seal the ends.

DNA Cloning Process

• Cutting DNA has to initially Use restriction enzymes at precise locations for generating fragmental vectors.
• Insertion is then induced in to a suitable vector (such as a plasmid) with a matching restriction enzyme to ensure ligation can occur.
• Transformation takes the vector and inserts into a host (such as bacteria)
• Selection and Idnetification helps determine whether the transformation included a recombinant DNA by using selectable marker to grow and test.

DNA Cloning

• DNA cloning inserts a specific DNA fragment into a vector for replication in a host organism, which Allows production of proteins from the cloned gene.

Requirements for Cloning

• Require replication system to create copies
• Require Vectors(Cloning kind):
  • A vector is required to transport the DNA fragment through cloning, and designed to replicate inside the host
    • Plasmids, bacteriophages, are kinds of cloning vectors
• Host System:
  • E. Coli can be used to replicate DNA.

Vecotrs and Host Systems

• Types: Host and Vectors
• Use Vectors and host according to size and fragment requirement.