Questions and Answers
What is methylation?
Methylation cannot eliminate the recognition sequence and it changes the sequence, making it unrecognizable and can't cut.
What are cloning vectors?
What is a genomic library?
A collection of plasmids or plasmids in bacteria that contain all cloned genes/entire human genome.
What did Khorana contribute to molecular biology?
Signup and view all the answers
Which method is used to make a radioactive gene probe?
Signup and view all the answers
What is a bacterial artificial chromosome (BAC)?
Signup and view all the answers
In the process of making recombinant DNA, EcoR1 is used to digest __________ DNA.
Signup and view all the answers
What is a palindromic sequence?
Signup and view all the answers
Sticky ends are easier to ligate than blunt ends.
Signup and view all the answers
What are the three main requirements for a cloning vector?
Signup and view all the answers
What is a selectable marker in cloning vectors?
Signup and view all the answers
What is PCR (polymerase chain reaction)?
Signup and view all the answers
What application does recombinant DNA technology have?
Signup and view all the answers
How is recombinant DNA protein produced from sheep?
Signup and view all the answers
What is the scheme for using phage lambda DNA as a cloning vector?
Signup and view all the answers
What are the applications of PCR?
Signup and view all the answers
How many SNPs are in the human genome?
Signup and view all the answers
What is RFLP analysis?
Signup and view all the answers
What is the process to see RFLP?
Signup and view all the answers
How could genetic researchers determine whether Akuada or her baby have sickle cell anemia?
Signup and view all the answers
What are the applications of biotechnology?
Signup and view all the answers
How are cloned genes used for producing products?
Signup and view all the answers
What are the necessary components of a PCR reaction?
Signup and view all the answers
Describe the process of Sanger sequencing.
Signup and view all the answers
What is Realtime PCR?
Signup and view all the answers
What is the utility of CRISPR/Cas9 technology?
Signup and view all the answers
List several genetically modified plants and the traits they possess.
Signup and view all the answers
Describe the function of the lacZ gene.
Signup and view all the answers
What is the significance of the polylinker in cloning vectors?
Signup and view all the answers
What happens during gene therapy for OTC deficiency?
Signup and view all the answers
What standard reagent is used in many therapeutic productions from genetic engineering?
Signup and view all the answers
A gene therapy for SCID caused leukemia in trials.
Signup and view all the answers
Gene therapy aims to correct disorders by inserting working copies of a gene into the __________ of a person.
Signup and view all the answers
Why doesn't a bacteria's restriction enzyme cut its own DNA?
Signup and view all the answers
What does blue white screening indicate?
Signup and view all the answers
What is a hazard or complication with somatic cell gene therapy?
Signup and view all the answers
What is DNA fingerprinting?
Signup and view all the answers
What does CRISPR stand for?
Signup and view all the answers
What are transgenic animals?
Signup and view all the answers
What is Cas9 nuclease?
Signup and view all the answers
What are the primers in PCR?
Signup and view all the answers
In a DNA digested by the restriction enzyme EcoRI, which fragment lengths are possible if the sequence specific to the probe is known?
Signup and view all the answers
What characterizes a polylinker region in a cloning plasmid?
Signup and view all the answers
Real-Time PCR involves gel analysis of the DNA produced.
Signup and view all the answers
How do you get DNA into a bacteria?
Signup and view all the answers
Why was human insulin not expressed after transformation into E. coli?
Signup and view all the answers
Cells harboring a plasmid with an intact lacZ gene form ___ colonies when grown on media with the β-galactosidase substrate X-gal, whereas cells with an interrupted lacZ gene form ___.
Signup and view all the answers
What are some applications of DNA technology?
Signup and view all the answers
What is a gene library?
Signup and view all the answers
What is library screening?
Signup and view all the answers
How are radioactive gene probes produced?
Signup and view all the answers
What is the average fragment length for EcoR1?
Signup and view all the answers
What percentage of the genome is used to make proteins?
Signup and view all the answers
What are some genetic variations in the genome?
Signup and view all the answers
What is Whole Exome Sequencing?
Signup and view all the answers
What is DNA sequence analysis?
Signup and view all the answers
What is the naming convention for restriction enzymes?
Signup and view all the answers
What is PCR?
Signup and view all the answers
What role does Taq polymerase have in PCR?
Signup and view all the answers
Who invented PCR?
Signup and view all the answers
Study Notes
Blue White Screening
- Involves cutting plasmid DNA and foreign DNA with the same restriction enzyme.
- Insertion of foreign DNA inactivates the lacZ gene, leading to differential colony color.
- Bacteria transformed with the recombinant plasmid become ampicillin resistant.
- White colonies indicate successful insertion of foreign DNA; blue colonies indicate no insertion.
- LacZ encodes β-galactosidase, which produces a blue pigment when no foreign DNA is present.
Somatic Cell Gene Therapy Complications
- Insertional mutations could occur, potentially activating nearby genes.
- Accessibility to target cells is a vital consideration for therapy effectiveness.
DNA Fingerprinting
- Utilizes analysis of DNA fragments as a method of identification.
- Techniques include Restriction Fragment Length Polymorphism (RFLP) and gel electrophoresis.
CRISPR
- Stands for Clustered Regularly Interspaced Short Palindromic Repeats.
- Provides bacteria with immunity against viruses, forming part of their adaptive immune system.
- Involves cutting sequences using the Cas9 enzyme guided by a synthesized RNA.
Transgenic Animals
- Organisms that incorporate genes transferred from different species, allowing for studies of gene function and production of proteins.
Cas9 Nuclease
- A bacterial enzyme that cuts double-stranded DNA at specified locations based on guide RNA.
PCR Primers
- Synthetic DNA oligonucleotides are essential for amplification processes.
Southern Blot Analysis
- Involves digesting genomic DNA with EcoRI and transferring fragments to a membrane for identification using probes.
- EcoRI is a restriction enzyme that helps segment DNA for identification purposes.
Polylinker Regions
- Characterized by multiple restriction cut sites facilitating the insertion of foreign DNA into plasmids for cloning purposes.
Real-Time PCR vs. Traditional PCR
- Real-Time PCR quantifies DNA during the amplification process and does not require gel analysis.
Transformation in Bacteria
- The process by which DNA is introduced into bacterial cells for genetic modifications.
Insulin cDNA Expression
- Expression failure can occur if the cDNA is not in the right reading frame or if posttranslational processing occurs differently in E. coli compared to eukaryotic cells.
Applications of DNA Technology
- Includes cancer treatment, insulin production, vaccine development, forensic science, and agricultural advancements like GMOs.
Gene Libraries
- Created by isolating and cloning DNA fragments into bacterial hosts, where each colony represents a unique gene.
Library Screening Techniques
- Involves transferring DNA from colonies to a membrane and probing for specific genes using labeled probes.
Producing Radioactive Gene Probes
- Involves using PCR and radioactive compounds to label specific DNA sequences for detection.
mRNA Extraction
- Achieved by utilizing the poly(A) tail present in mRNA molecules.
cDNA Synthesis
- Involves reverse transcription of mRNA using oligo(dT) primers, producing DNA without introns.
NGS - Next Generation Sequencing
- New sequencing techniques that offer faster and cost-effective DNA analysis compared to traditional methods.
SNP's and Genetic Variations
- Single Nucleotide Polymorphisms (SNPs) are the most common type of genetic variation, significant for disease studies.
Protein Sequencing
- Involves isolating proteins and raising antibodies; however, redundancies in the genetic code present challenges.
Applications of PCR
- Effective in amplifying small samples for various analyses including prenatal diagnosis and forensic applications.
RFLP Analysis
- Detects genetic variations that alter fragment sizes or recognition sites, aiding diagnostic processes for genetic disorders.
Sickle Cell Anemia Testing
- Can utilize Southern blot techniques to determine if mutations indicative of sickle cell anemia are present in samples.
Biotechnological Applications
- Includes the use of RFLPs in processes like fermentation, brewing, and treatment of wastewater.
Cloned Genes for Product Production
- Genes can be introduced into bacteria to produce desired proteins if proper regulatory sequences are present.### Cloning and Gene Expression
- Bacteria transcribe and synthesize proteins from cloned genes, facilitating the expression of foreign genes.
- Examples of proteins produced include human insulin, interferon, somatostatin (growth hormone), and enzymes like DNA polymerase.
Gene Introduction and Transformation
- Genes can be introduced into higher organisms, such as animals and plants, via methods like viral transformation.
- Transforming mice with growth hormone genes creates "supermice."
- Plants are subjected to transformation, often using Agrobacterium, creating traits like herbicide resistance to glyphosate and resistance to viral diseases.
Multiple-Cloning Sites and Expression Vectors
- Multiple-cloning sites (MCS) can have up to 20 unique restriction sites, facilitating DNA insertion.
- MCS in expression vectors is positioned downstream of the promoter, enabling the production of protein products such as insulin, vaccines, antibiotics, and gene therapies.
Blue-White Screening and the lacZ Gene
- The lacZ gene encodes β-galactosidase, which can metabolize x-gal to produce a blue pigment.
- A mutation in lacZ can create a white colony, indicating successful DNA uptake, while blue indicates no DNA was integrated.
PCR Fundamentals
- Key components of a PCR reaction include DNA polymerase, primers, deoxynucleotide triphosphates (dNTPs), and template DNA.
- PCR temperature cycling involves denaturation (95°C), primer annealing (55-65°C), and extension (72°C).
Sanger Sequencing
- Target DNA is repeatedly copied, creating fragments of varying lengths marked by fluorescent dye-labeled dideoxy nucleotides that terminate the chain.
- The process enables sequencing by revealing the specific nucleotide sequence based on color coding.
Realtime PCR
- Utilizes a thermocycler for temperature cycling processes: denaturation, annealing, and extension.
- It offers high sensitivity, efficiency, and precise quantitation of DNA.
Nucleic Acid Quantification
- Techniques include northern blotting, microarrays, and realtime PCR, with realtime PCR being the most sensitive and quantitative.
Genetic Fingerprinting with VNTRs
- Alec Jeffreys pioneered the use of hypervariable minisatellites for DNA fingerprinting.
- The process involves cutting DNA, electrophoresis, blotting, and hybridization with a radioactive probe to create unique banding patterns.
pBR322 and pUC Cloning Vectors
- Digesting pBR322 with EcoRI allows for recombinant DNA cloning; however, the plasmid can re-circularize.
- pUC18 and pUC19 vectors help distinguish recombinant clones through a blue-white screening, with advantages in cloning site accessibility.
CRISPR/Cas9 Technology
- A gene-editing tool holding promise for correcting genetic diseases and modifying agricultural traits by altering existing genes in crops.
Electroporation and RNA Interference
- Electroporation introduces recombinant DNA into cells through temporary membrane disruption caused by electrical pulses.
- RNA interference inhibits the action of messenger RNA, providing a temporary solution compared to CRISPR's permanent gene inactivation.
Applications in Agricultural Biotechnology
- Transgenic crops include BT corn with genes from Bacillus thuringiensis for pest resistance and golden rice enriched with beta carotene and iron for enhanced nutrition.
Gene Therapy
- Aims to correct genetic disorders by introducing functional gene copies into patients’ cells.
- Notable examples include ADA deficiency and cystic fibrosis, with challenges presented in trials, like SCID leading to leukemia in some instances.
Cloning Vectors
- Different types of cloning vectors include plasmids, phages, BACs (bacterial artificial chromosomes), and YACs (yeast artificial chromosomes), each with varying carrying capacities for foreign DNA.
Genetic Engineering and DNA Probes
- Techniques like RFLP analysis are employed for livestock and crop breeding; specific probes can hybridize to detect unique DNA sequences across individuals.
Summary of Techniques
- Techniques such as hybridization, PCR, and cloning vectors are foundational to genetic engineering and biotechnology, driving advances in medicine, agriculture, and research.### pZErO Plasmid
- Produces a protein that kills bacteria that do not possess the plasmid.
- Bacteria survives if it incorporates a plasmid with an insert.
- No colonies form without an insert due to the presence of two special open reading frames (ORFs).
- Absence of inserted DNA leads to expression of lac z and ccdb genes; ccdb gene results in a toxin that inhibits E. coli replication, causing cell death.
- Presence of inserted DNA disrupts the expression of lac z and ccdb, preventing E. coli cell death.
PCR (Polymerase Chain Reaction)
- Allows amplification of specific DNA regions from extremely small amounts, producing millions of copies.
- Serves as a foundational technique in molecular biology for sequencing, cloning, and genetic manipulation.
- Requires template DNA, two primers, dideoxynucleotides, and thermostable DNA polymerase (Taq) for amplification.
- Only trace amounts of the template DNA are required, making it valuable for diagnostics and forensics.
- Most accurate method for analyzing mRNA expression, although it can be costly.
Uses of Cloned Genes (Applications of Recombinant DNA Technology)
- Production of proteins through microbes for pharmaceutical uses, such as human growth hormone, insulin, and TPA (tissue plasminogen activator).
- Implementation of gene therapy to correct human genetic diseases.
Producing Recombinant DNA Protein from Sheep
- Utilizes plasmids with a specific promoter (galactoglobulin) that is only expressed in mammary glands.
- Cloning involves injecting a sheep egg with a plasmid; the DNA integrates into the sheep's chromosomes.
- Fertilization occurs in vitro, allowing the sheep to grow while carrying the genetic construct in its genome.
- The protein is secreted into the sheep's milk, which contains fewer proteins, facilitating easier purification.
Scheme for Using Phage Lambda DNA as a Cloning Vector
- Lambda DNA is modified to retain only the necessary left and right arms for phage functionality, with internal genes removed.
- Left and right arms of lambda chromosomes are procured for cloning purposes.
- Insert DNA is added and ligated using EcoR1 to facilitate the incorporation of DNA.
- Human DNA can be inserted into the phage DNA, which self-assembles when phage proteins are pipetted in.
- The resulting bacteriophage infects E. coli, integrating the DNA, which E. coli then replicates, allowing for the generation of larger DNA fragments (up to 20kb) without relying heavily on transformation techniques.
Studying That Suits You
Use AI to generate personalized quizzes and flashcards to suit your learning preferences.
Description
Prepare for your Genetics Exam with these comprehensive flashcards covering Chapters 8 and 10. Each card includes essential terms and concepts related to genetics. Test your knowledge on topics such as blue-white screening and its applications in molecular biology.