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Expose the membrane to UV to crosslink DNA to the blot, incubate with radioactive probe, wash blot to remove excess probe, expose to X-ray film (autoradiography), bands on film represent sites where the probe annealed
DNA blotting:
In vitro amplification of specific DNA fragments, relies on temperature changes to promote denaturation of dsDNA, annealing of primers, and polymerization of new DNA
Polymerase Chain Reaction (PCR):
Method where RNA is first converted to cDNA and then the cDNA is amplified by PCR, sensitive technique for detecting and quantifying mRNA
Reverse transcription – PCR (RT-PCR):
PCR method for measuring the increase in the amount of DNA as it is amplified, can provide accurate quantification of mRNA levels in a sample
Real-time PCR (real-time quantitative PCR):
Technology that uses next-generation sequencing to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time
RNA-seq (RNA sequencing):
Frequent differences in DNA sequence between individuals, including SNPs, STRs, and VNTRs
DNA Polymorphisms:
Base pair differences between individuals, most are in non-coding regions, can affect gene function if in regulatory regions
Single Nucleotide Polymorphisms (SNPs):
Detect SNPs by using restriction enzymes, as SNPs may create or abolish restriction sites
Restriction Fragment Length Polymorphisms (RFLPs):
Detect SNPs by using allele-specific oligonucleotides that hybridize to the target sequence
Allele-specific oligonucleotide hybridization (ASO):
Experiment that uses a DNA microarray to study gene expression patterns
DNA microarray:
Very short tandem repeats (2-6 bp), amplified by PCR with primers flanking the repeats
Short Tandem Repeats (STRs):
Longer tandem repeats (7 bp or more), detected by restriction digestion and Southern blotting
Variable Number Tandem Repeats (VNTRs):
Genetic testing that focuses on the molecular nature of mutations associated with disease
DNA Molecular Testing for Human Genetic Disease Mutants:
Examples of genetic diseases that can be tested for using DNA molecular testing
Huntington disease, Hemophilia, Cystic fibrosis, Tay-Sachs disease, Sickle-cell anemia, Down syndrome, Breast cancer:
Genes that control cell growth in breast and ovarian tissues, mutations in these genes can lead to breast cancer
BRCA1 and BRCA2 genes:
Use of RFLP marker information as DNA markers, heterozygotes show both parental types
Restriction Fragment Length Polymorphism (RFLP) analysis:
Detection of the sickle-cell gene mutation using DdeI restriction fragment length polymorphism
Sickle-cell gene detection:
Technique used to detect mutations in the open-angle glaucoma gene GLC1A
Allele-specific oligonucleotide (ASO) hybridization:
Technique used to identify the location of a gene responsible for a genetic disorder
Chromosome walking (Positional Cloning):
Marker-assisted molecular technique to identify individuals based on their DNA profiles
DNA Typing (DNA fingerprinting or DNA Profiling):
Forensic science, paternity and maternity testing, population studies, proving horse pedigree, conservation biology, detection of pathogenic E. coli, detection of genetically modified organisms, historical questions
Applications of DNA Typing:
To identify specific DNA sequences on a membrane by hybridizing a radioactive probe
What is the purpose of DNA blotting?
Denaturation of dsDNA, annealing of primers, and polymerization of new DNA
What are the main steps in the Polymerase Chain Reaction (PCR)?
RNA is first converted to cDNA, which is then amplified by PCR
How does Reverse Transcription-PCR (RT-PCR) work?
To measure the increase in DNA amount during amplification, allowing for accurate quantification of mRNA levels
What is the purpose of Real-time PCR?
SNPs are single base pair differences, STRs are short tandem repeats, VNTRs are longer tandem repeats
What is the difference between SNPs, STRs, and VNTRs?
SNPs may create or abolish restriction sites, leading to different restriction fragment lengths that can be detected
How are RFLPs used to detect SNPs?
Using oligonucleotides that are specific to the allele of interest to detect the presence of that allele
What is the principle behind Allele-Specific Oligonucleotide (ASO) hybridization?
Gene expression patterns across multiple genes or samples can be studied
What information can be obtained from a DNA microarray experiment?
STRs are amplified by PCR with primers flanking the repeats, and the fragment lengths are analyzed
How are STRs detected and analyzed?
VNTRs are detected by restriction digestion and Southern blotting, as they are too long to be amplified by PCR
How are VNTRs detected?
To determine if an individual has a specific gene mutation associated with a genetic disease
What is the purpose of DNA molecular testing for genetic diseases?
Huntington disease, Hemophilia, Cystic fibrosis, Tay-Sachs disease, Sickle-cell anemia, Down syndrome, Breast cancer
What are some examples of genetic diseases that can be tested using DNA molecular methods?
Mutations in these genes can lead to uncontrolled cell growth in breast and ovarian tissues
How are mutations in the BRCA1 and BRCA2 genes related to breast cancer?
RFLP markers can be used to identify individuals based on their unique restriction fragment patterns
What is the principle behind RFLP analysis?
The sickle-cell mutation creates or abolishes a restriction site, leading to different fragment lengths
How is the sickle-cell gene mutation detected using RFLP analysis?
To identify the location of a gene responsible for a genetic disorder
What is the purpose of chromosome walking (positional cloning)?
To identify individuals based on their unique DNA profiles
What is the general purpose of DNA typing (DNA fingerprinting or DNA profiling)?
Forensic science, paternity/maternity testing, population studies, conservation biology, detection of pathogenic bacteria, detection of GMOs, historical questions
What are some applications of DNA typing?
DNA blotting is used to identify specific DNA sequences, while DNA typing is used to identify individuals based on their unique DNA profiles
What is the difference between DNA blotting and DNA typing?
Here are 70 flashcards based on the extensive content provided on genomics, genetic engineering, gene therapy, and CRISPR technology:
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What is genetic engineering?
Genetic engineering is the process of producing modified DNA in vitro and introducing recombinant DNA into host organisms.
What are model organisms in genetic engineering?
Yeast, plants, and mice are commonly used model organisms for genetic engineering.
What is gene disruption in yeast?
Gene disruption involves inactivating a gene to study its function, often by replacing it with a selectable marker in yeast.
What is the yeast two-hybrid system?
A technique to study protein-protein interactions in yeast by fusing proteins of interest with a binding domain and an activation domain.
What are some commercial products of biotechnology?
Products include insulin, human growth hormone, vaccines, and genetically modified crops.
What is gene therapy?
Gene therapy is a technique to correct defective genes responsible for disease development by inserting functional genes into patients.
What is a vector in gene therapy?
A vector is a carrier molecule used to deliver a therapeutic gene to target cells, commonly using modified viruses.
What are retroviruses used for in gene therapy?
Retroviruses can insert double-stranded DNA copies of their RNA genomes into the host genome, making them useful for gene therapy.
What are adenoviruses used for?
Adenoviruses, which cause respiratory infections, are used as vectors in gene therapy to deliver therapeutic genes.
What is adeno-associated virus (AAV) used for?
AAV is a small virus used in gene therapy to insert genetic material into a specific site on chromosome 19.
What was a major setback in gene therapy in 1999?
The death of Jesse Gelsinger during a gene therapy trial led to increased safety regulations.
What challenges has gene therapy faced?
Challenges include immune responses, short-lived therapy effects, issues with viral vectors, and difficulty treating multigene disorders.
What breakthrough occurred in gene therapy for blindness in 2008?
Gene therapy improved sight in patients with Leber’s congenital amaurosis by inserting a functional RPE65 gene.
What disease did gene therapy halt in two boys in 2009?
Gene therapy halted Adrenoleukodystrophy (ALD) using a modified HIV virus to deliver a therapeutic gene.
What is ALD (Adrenoleukodystrophy)?
ALD is a genetic disorder caused by a defective ABCD1 gene, leading to fat buildup and nerve damage, primarily affecting boys.
What is Tay-Sachs disease?
A genetic disorder that causes brain damage due to a lack of enzyme Hex-A, leading to motor and mental deterioration in children.
What is CRISPR-Cas9?
A genome-editing technology that uses the Cas9 enzyme guided by RNA to make precise cuts in DNA for gene modification.
What are zinc-finger nucleases (ZFNs)?
ZFNs are engineered proteins used to make specific cuts in DNA, enabling targeted genome editing.
What are TALENs?
Transcription Activator-Like Effector Nucleases (TALENs) are proteins used for precise gene editing by making targeted DNA cuts.
What is a genomic library?
A collection of DNA fragments representing an organism’s entire genome, stored in vectors for study and replication.
What is a cDNA library?
A collection of DNA synthesized from mRNA, representing only the expressed genes in a particular cell or tissue type.
What is PCR?
Polymerase Chain Reaction (PCR) is a technique used to amplify specific DNA sequences, producing millions of copies.
What is the purpose of a restriction enzyme?
Restriction enzymes cut DNA at specific sites, creating fragments used in cloning and DNA analysis.
What are sticky and blunt ends in DNA?
Sticky ends have overhanging nucleotides, while blunt ends are straight cuts
both are products of restriction enzyme digestion.
What is Southern blotting?
A method to detect specific DNA sequences by transferring DNA from a gel to a membrane and probing with labeled DNA.
What is Northern blotting?
Northern blotting is used to detect specific RNA sequences by hybridizing RNA with a complementary DNA probe.
What is Western blotting?
Western blotting is used to detect specific proteins in a sample using antibodies that bind to target proteins.
What is Sanger sequencing?
A DNA sequencing method using chain-terminating nucleotides to determine the order of bases in DNA.
What is next-generation sequencing?
A high-throughput DNA sequencing technology that allows simultaneous sequencing of millions of DNA fragments.
What is an origin of replication in plasmids?
The origin of replication is a sequence that enables a plasmid to replicate independently within a host cell.
What is a selectable marker in cloning?
A gene in a plasmid that allows cells with the plasmid to survive under selective conditions, confirming successful cloning.
What is gene cloning?
Gene cloning is the process of making multiple copies of a gene by inserting it into a vector and growing it in host cells.
What is a polylinker or multiple cloning site (MCS)?
An MCS is a short DNA segment with multiple restriction sites, allowing insertion of DNA fragments during cloning.
What is a reporter gene?
A reporter gene produces a measurable product, such as a fluorescent protein, indicating successful gene expression in cells.
What is a genomic equivalent?
The number of clones needed in a library to cover an entire genome at least once.
What is a transformation in cloning?
Transformation is the process of introducing recombinant DNA into host cells, often using heat shock or electroporation.
What are BACs and YACs?
Bacterial Artificial Chromosomes (BACs) and Yeast Artificial Chromosomes (YACs) are large cloning vectors used to carry large DNA fragments.
What is colony hybridization?
A technique to identify bacterial colonies containing a specific DNA sequence using a labeled DNA probe.
What is a clone?
A genetically identical copy of a DNA fragment, cell, or organism, produced through techniques like cloning or cell division.
What are plasmids?
Small, circular DNA molecules used as vectors for cloning and gene expression in bacterial cells.
What is electroporation?
A technique to introduce DNA into cells by applying an electric field to increase cell membrane permeability.
What is transfection?
The process of introducing nucleic acids into eukaryotic cells, often using chemical or physical methods.
What is a probe in molecular biology?
A labeled DNA or RNA sequence used to detect complementary sequences in DNA or RNA samples.
What is gene therapy’s goal?
The goal is to treat or prevent disease by inserting, modifying, or correcting defective genes in patients.
What is a viral vector?
A modified virus used to deliver therapeutic genes in gene therapy, commonly used include retroviruses and adenoviruses.
What is a knockout mouse?
A genetically engineered mouse in which a specific gene has been inactivated or "knocked out" to study its function.
What is the CRISPR-Cas9 mechanism?
CRISPR uses a guide RNA to target DNA, while Cas9 makes a double-strand break, allowing for targeted gene editing.
What is a restriction fragment length polymorphism (RFLP)?
A variation in DNA fragment sizes caused by mutations at restriction enzyme sites, used for genetic analysis.
What is a transgenic organism?
An organism with a foreign gene inserted into its genome, commonly used in research and agriculture.
What is a gene drive?
A genetic engineering technique that spreads a specific gene throughout a population by biasing inheritance patterns.
What are antisense oligonucleotides?
Short DNA or RNA molecules that bind to mRNA to inhibit translation and regulate gene expression.
What is gene knockout?
Gene knockout is a technique to inactivate or delete a gene to study its function in an organism.
What is in vitro fertilization (IVF)?
A process of fertilizing an egg outside the body and implanting it in the uterus, sometimes using genetically modified embryos.
What is homologous recombination?
A process where DNA sequences exchange between two similar or identical DNA molecules, used in gene targeting.
What is a chimera in genetics?
An organism with cells from two different zygotes, often used in research to study gene function.
What is a gene gun?
A device used to insert DNA-coated particles into plant cells, a common tool for creating genetically modified plants.
What is RNA interference (RNAi)?
A process by which RNA molecules inhibit gene expression by destroying mRNA molecules, regulating gene activity.
What is a knockout library?
A collection of organisms or cells where each has a different gene inactivated, used for functional genomics.
What is an inducible promoter?
A promoter that initiates gene expression only in the presence of a specific inducer or condition.
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