Genetics Exam 2: Chapters 8, 10 Flashcards

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?

pZErO plasmid

Plasmids

BACs

All of the above (correct)

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?

Khorana found the identity of triplets and figured out the genetic code by identifying STOP codons and a START codon. Signup and view all the answers

Which method is used to make a radioactive gene probe?

A and C Signup and view all the answers

What is a bacterial artificial chromosome (BAC)?

An artificial version of a bacterial chromosome that can carry inserts of 100,000 to 500,000 base pairs. Signup and view all the answers

In the process of making recombinant DNA, EcoR1 is used to digest __________ DNA.

human Signup and view all the answers

What is a palindromic sequence?

DNA sequence that is identical when read forwards and backwards Signup and view all the answers

Sticky ends are easier to ligate than blunt ends.

True Signup and view all the answers

What are the three main requirements for a cloning vector?

Selectable marker, cloning site (EcoR1 site), origin of replication. Signup and view all the answers

What is a selectable marker in cloning vectors?

A gene introduced into a cell that confers a trait suitable for artificial selection, such as antibiotic resistance. Signup and view all the answers

What is PCR (polymerase chain reaction)?

A technique to amplify specific regions of DNA, creating millions of copies from small amounts. Signup and view all the answers

What application does recombinant DNA technology have?

Both A and B Signup and view all the answers

How is recombinant DNA protein produced from sheep?

A plasmid with a special promoter is used, and the gene is injected into a sheep egg. Signup and view all the answers

What is the scheme for using phage lambda DNA as a cloning vector?

Phage lambda DNA is modified, and after inserting human DNA, it self-assembles and infects E.coli. Signup and view all the answers

What are the applications of PCR?

Cell free cloning, amplification of small samples, prenatal diagnosis, forensic analysis, DNA sequencing. Signup and view all the answers

How many SNPs are in the human genome?

3 million Signup and view all the answers

What is RFLP analysis?

Restriction fragment length polymorphism, a technique to detect differences in DNA sequences. Signup and view all the answers

What is the process to see RFLP?

Isolate DNA, cut using a restriction enzyme, run gel, and perform a southern blot. Signup and view all the answers

How could genetic researchers determine whether Akuada or her baby have sickle cell anemia?

Use a Southern blot and run on a gel. Signup and view all the answers

What are the applications of biotechnology?

Brewing, antibiotics, biological fermentations, sewage treatment, and wine making. Signup and view all the answers

How are cloned genes used for producing products?

Introduce the gene into bacteria or higher organisms for protein synthesis. Signup and view all the answers

What are the necessary components of a PCR reaction?

DNA polymerase, primers, dNTPs, template. Signup and view all the answers

Describe the process of Sanger sequencing.

Target DNA is copied with chain-terminator nucleotides marking the ends of fragments. Signup and view all the answers

What is Realtime PCR?

A kinetic approach using a thermocycler for more efficient and sensitive data collection. Signup and view all the answers

What is the utility of CRISPR/Cas9 technology?

Used for genomic surgery to correct genes causing diseases. Signup and view all the answers

List several genetically modified plants and the traits they possess.

Crops with Bt toxin for insect resistance and Golden Rice for added nutritional value. Signup and view all the answers

Describe the function of the lacZ gene.

Encodes for β-galactosidase which can break down x-gal into a blue pigment. Signup and view all the answers

What is the significance of the polylinker in cloning vectors?

Allows DNA to be inserted into a specific region of the plasmid. Signup and view all the answers

What happens during gene therapy for OTC deficiency?

Normal OTC gene is inserted to correct the deficiency, preventing ammonia buildup. Signup and view all the answers

What standard reagent is used in many therapeutic productions from genetic engineering?

Human insulin. Signup and view all the answers

A gene therapy for SCID caused leukemia in trials.

True Signup and view all the answers

Gene therapy aims to correct disorders by inserting working copies of a gene into the __________ of a person.

cells Signup and view all the answers

Why doesn't a bacteria's restriction enzyme cut its own DNA?

Because it is modified or methylated. Signup and view all the answers

What does blue white screening indicate?

White colonies contain foreign DNA. Signup and view all the answers

What is a hazard or complication with somatic cell gene therapy?

Insertional mutation possible Signup and view all the answers

What is DNA fingerprinting?

Analysis of fragments of DNA as a form of identification Signup and view all the answers

What does CRISPR stand for?

Clustered regularly interspaced short palindromic repeats Signup and view all the answers

What are transgenic animals?

Animals that contain genes transferred from other animals, usually from a different species Signup and view all the answers

What is Cas9 nuclease?

A bacterial endonuclease that cuts double-stranded DNA at a location determined by a segment of guide RNA Signup and view all the answers

What are the primers in PCR?

Synthetic DNA oligonucleotides 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?

2 kb, 4.0-kb DNA fragment Signup and view all the answers

What characterizes a polylinker region in a cloning plasmid?

Multiple restriction cut sites Signup and view all the answers

Real-Time PCR involves gel analysis of the DNA produced.

False Signup and view all the answers

How do you get DNA into a bacteria?

Transformation into the bacteria Signup and view all the answers

Why was human insulin not expressed after transformation into E. coli?

The cDNA must be inserted in the correct reading frame. 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 _.

blue, white Signup and view all the answers

What are some applications of DNA technology?

Production of insulin Signup and view all the answers

What is a gene library?

A collection of all bacteria containing cloned genes Signup and view all the answers

What is library screening?

A method for choosing colonies containing desired genes using probes Signup and view all the answers

How are radioactive gene probes produced?

By annealing hexanucleotide random primers and extending with Klenow fragment in the presence of radioactive precursor Signup and view all the answers

What is the average fragment length for EcoR1?

(4)^6 - 6 base cutter - 4096 base pairs Signup and view all the answers

What percentage of the genome is used to make proteins?

1-2% Signup and view all the answers

What are some genetic variations in the genome?

SNPs, indels, copy number variants, block substitution, inversion variant Signup and view all the answers

What is Whole Exome Sequencing?

Method for determining the precise order of nucleotide bases in the exome Signup and view all the answers

What is DNA sequence analysis?

Methods including the Sanger method and Next Generation Sequencing Signup and view all the answers

What is the naming convention for restriction enzymes?

Based on the bacterium from which it was isolated Signup and view all the answers

What is PCR?

Enzymatic amplification of a DNA fragment flanked by two oligonucleotide primers Signup and view all the answers

What role does Taq polymerase have in PCR?

It is a heat-resistant DNA polymerase used for amplifying DNA Signup and view all the answers

Who invented PCR?

Kary Mullis 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.

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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.