Chapter 10 : Gene isolation AND manipulation

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Questions and Answers

What is the primary purpose of dideoxynucleotides (ddNTPs) in Sanger sequencing?

To enhance the speed of DNA synthesis

To terminate chain elongation during DNA synthesis (correct)

To increase the yield of PCR products

To selectively amplify specific DNA regions

In the context of genome sequencing, what does assembling contigs involve?

Extracting specific genes from genomic DNA

Creating a full genome from a single long DNA sequence

Identifying mutations by comparing multiple genomes

Linking overlapping sequence reads to form continuous sequences (correct)

Which of the following best describes the process of Whole Genome Shotgun Sequencing (WGS)?

Cloning fragments into microbial hosts for amplification

Sequencing individual fragments and then constructing a genome from the data (correct)

Sequencing only specific regions of the genome

Mapping the genome before sequencing individual fragments

What is a primary advantage of Next Generation Sequencing (NGS) compared to traditional sequencing methods?

It allows for simultaneous sequencing of millions of DNA fragments Signup and view all the answers

How are sequence reads typically detected in fluorescent Sanger sequencing?

Through a laser detection system connected to a computer Signup and view all the answers

What percentage of the human genome is responsible for encoding protein products?

1.5% Signup and view all the answers

What is defined as a paralog?

Genes that arise from gene duplication events. Signup and view all the answers

What type of gene remains functional in the platypus regarding yolk proteins?

Functional yolk gene. Signup and view all the answers

How many pseudogenes exist in the human genome?

19,000 Signup and view all the answers

What method is used to identify transcription factor binding sites?

Chromatin Immunoprecipitation (ChIP) Signup and view all the answers

What is the main reason for increased complexity in humans compared to other species?

Gene duplications leading to variation in protein functions. Signup and view all the answers

What percentage of open reading frames (ORFs) in humans have no known function?

42% Signup and view all the answers

How many splice variants do 60% of human genes have on average?

3 Signup and view all the answers

What is the primary purpose of the yeast two-hybrid system?

To investigate protein-protein interactions Signup and view all the answers

Which gene in pathogenic E. coli O157:H7 is responsible for its unique virulence factors?

Unique genes in O157:H7 Signup and view all the answers

What happens when the bait protein and target proteins interact in the yeast two-hybrid system?

They activate the Gal4 promoter and initiate transcription Signup and view all the answers

In comparative genomics, what distinguishes pathogenic E. coli O157:H7 from non-pathogenic K12?

O157:H7 has unique genes associated with toxins and adhesion Signup and view all the answers

What is a consequence of the adherence proteins present in pathogenic E. coli O157:H7?

Modification of membrane structure leading to intestinal damage Signup and view all the answers

What is the first step in the gene cloning process?

Extract the gene of interest from DNA Signup and view all the answers

Which enzyme is responsible for joining DNA fragments during cloning?

Ligase Signup and view all the answers

How does EcoRI behave when it cuts DNA?

Produces sticky ends with single-stranded overhangs Signup and view all the answers

What is the purpose of a selectable marker in plasmid cloning?

To identify successful transformation in bacteria Signup and view all the answers

Which of these describes the polymerase chain reaction (PCR)?

An in vitro process for amplifying specific DNA sequences Signup and view all the answers

What distinguishes a blue colony from a white colony in colony screening?

Blue colonies are a result of X-gal cleavage; white colonies are not Signup and view all the answers

Which of the following statements about Taq polymerase is accurate?

It is error-prone but commonly used for high-temperature PCR Signup and view all the answers

What is the significance of using two different restriction enzymes in cloning?

To control the directionality of ligation Signup and view all the answers

What is the primary purpose of adding a His-tag to a protein?

To facilitate purification using Ni2+-coated beads Signup and view all the answers

Which method is NOT used for delivering recombinant DNA into bacterial cells?

Electroporation with BACs Signup and view all the answers

What technique uses EcoRI digestion to confirm the presence of a gene insert?

Agarose gel analysis Signup and view all the answers

Which of the following best describes Gibson Assembly?

It assembles multiple DNA fragments using overlapping ends Signup and view all the answers

In Southern Blotting, what is the primary purpose of using a labeled probe?

To detect the presence of specific DNA sequences Signup and view all the answers

Dideoxy Sequencing is known for using which type of nucleotide to terminate chain elongation?

Dideoxynucleotide triphosphates Signup and view all the answers

Which cloning technique allows for the assembly of DNA fragments without creating sequence scars?

Seamless Cloning Signup and view all the answers

What does the T5 exonuclease do during Gibson Assembly?

Creates overlapping ends by chewing back DNA Signup and view all the answers

What temperature is used to denature DNA during PCR?

95°C Signup and view all the answers

Which polymerase is primarily used during the extension phase of PCR?

Taq polymerase Signup and view all the answers

Why is it necessary to ensure that the target sequence does not contain an EcoRI site during PCR product digestion?

To avoid cleavage of the fragment of interest Signup and view all the answers

What component is included in the forward primer for in vitro transcription using T7 polymerase?

T7 promoter sequence Signup and view all the answers

What is the purpose of using reverse transcriptase in the process of making cDNA?

To convert mRNA into cDNA Signup and view all the answers

When preparing cDNA, what is the role of oligo-dT primers?

To hybridize with the poly-A tail of mRNA Signup and view all the answers

What is the purpose of adding sticky ends to cDNA before inserting it into a plasmid vector?

To allow for specific binding during ligation Signup and view all the answers

How does IPTG affect the process of expressing eukaryotic genes in bacteria?

It activates T7 polymerase for transcription Signup and view all the answers

What is the purpose of using ganciclovir in the selection process of cells with targeted gene knockouts?

To kill cells that have not undergone the knockout. Signup and view all the answers

What is one limitation of ESTs when identifying gene structures?

They often miss the full length of the gene due to splicing. Signup and view all the answers

Which of the following steps is involved in producing a mouse with a targeted gene knockout?

Injecting selected cells into blastocyst stage embryos. Signup and view all the answers

What does RNAseq allow researchers to measure in a sample?

The quantified gene expression levels. Signup and view all the answers

In the context of gene expression, what does a higher number of reads in RNAseq generally indicate?

Higher expression level of the gene. Signup and view all the answers

What is the primary role of the Basic Local Alignment Search Tool (BLAST)?

To evaluate the homology of gene sequences. Signup and view all the answers

What ratio of genotypes can be expected from the offspring when mating a chimera with a black mouse?

1:2:1 Signup and view all the answers

What does digital drop PCR specifically measure in relation to gene expression?

Accuracy in measuring mRNA concentration. Signup and view all the answers

What is the primary purpose of using microinjection in genetic engineering?

To introduce DNA directly into cells Signup and view all the answers

Which method uses Agrobacterium tumefaciens for gene insertion in plants?

Ti plasmid modification Signup and view all the answers

What is a key feature of homologous recombination in gene targeting?

Specific targeting of a mutated gene Signup and view all the answers

What is one possible outcome of targeted insertion using homologous recombination?

Ectopic insertion of the neomycin resistance marker Signup and view all the answers

What is the mechanism by which the T-DNA integrates into the plant's chromosome during transformation?

Natural infection and genetic modification Signup and view all the answers

What does a single crossover event in homologous recombination achieve?

Corrects the gene but retains the mutant gene Signup and view all the answers

What is the expected segregation pattern of progeny when T-DNA is inserted into one chromosome pair?

1:2:1 ratio Signup and view all the answers

In the context of virus infection for genetic delivery, what is one role of saponin?

To enhance the immune response Signup and view all the answers

What characterizes the outcome labeled as 'No Insertion' in homologous recombination?

Neomycin sensitive with no change Signup and view all the answers

What does the method of creating transgenic plants primarily aim to achieve?

To insert specific genes into plant cells Signup and view all the answers

Study Notes

Sanger Sequencing Steps

DNA template (PCR product, plasmid insert, genomic/cDNA).
Primer (3' OH for DNA synthesis).
Normal dNTPs (A, T, C, G).
A small amount of dideoxynucleotide (ddNTP: A, T, C, or G).

Reaction

DNA synthesis with dNTP incorporated at random, terminating chain elongation.
Separated by size on a gel (smaller fragments run further).
DNA sequence read from the bottom (smallest fragment) to the top (longest fragment).

Reading

Fluorescent ddNTPs: Use of fluorescently labeled ddNTPs (green, red, blue, yellow for A, T, C, G).
Sequence read from electrophoresis, detected by a laser and recorded by computer.

Genome Sequencing Process

Fragmenting Genome

Cut the genome into random fragments and sequence each fragment.

Overlap & Contig

Overlap sequence reads to assemble fragments (continuous sequences).
Method developed by Craig Venter (founder of Celera Genomics, sequenced human genome).

Whole Genome Shotgun Sequencing (WGS)

Sequence first, map later.

Paired-End Reads

Sequence both ends of a fragment to help assemble larger genomes.
End reads from multiple clones overlap to create a full genome.

Next Generation Sequencing (NGS)

Example: Illumina dye sequencing (many systems available).

Key Features

Sequencing without cloning into microbial hosts.
Parallel sequencing of millions of DNA fragments.
Short reads (<100 base pairs).

DNA Library Construction:

DNA is fragmented, ends are repaired (blunt), and adaptors are added.

DNA Fragment Binding:

Fragments bind to flow cells and undergo bridge amplification.

Sequencing by Synthesis:

DNA synthesis incorporates fluorescently labeled nucleotides.

Cycle Repeats:

DNA synthesis incorporates its complementary adaptor on the slide.
Reads are aligned to a reference genome using bioinformatics tools.

NGS struggles with repetitive sequences and gaps in the genome

Solution - Circularized Fragments: Circular DNA libraries can capture end sequences and fill gaps.

Genetic Engineering

Methods: Introduce a gene (transgene) into an organism (transgenic organism).

Transformation

Making cells competent to take up plasmid DNA.

Liposome Fusion:

RNA vaccines (e.g., Pfizer COVID-19 vaccine) delivered through lipid bilayers.

Electroporation:

Electrical pulses create holes in membranes, allowing DNA entry.

Biolitic Delivery (Gene Gun):

Metal-coated beads shot into plant cells.

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Description

Test your knowledge on key concepts in genomics and various sequencing techniques, including Sanger sequencing, Next Generation Sequencing, and genome assembly. This quiz covers critical aspects such as the role of ddNTPs, the definition of paralogs, and the complexity of the human genome.