Intro Molecular Genetics Exam Three

Describe the necessary features of shuttle vectors

→Origin for species 1

→Selectable marker for species 1

→Origin for species 2

→Selectable marker for species 1

→Multiple cloning site

Describe the induction/repression of the lacUV and tet promoter

Components of lacUV

1. lac promoter (lacUV varient)

2. LacI repressor

3. Isopropyl B-d-1-thiogalactopyranoside (IPTG) is an inducer

Default state if OFF. When you add IPTG it turns on. Very useful when you want inducible expression within bacteria

Tet system

1. Tet-Off system: tetracycline prevents the tTA transcription factor from binding at the promoter. Gene expression is inhibited in the presence of tetracycline

2. Tet-On system: Tetracycline binds the rtTA transcription factor and allows it to bind DNA at the promoter. Gene expression is induced in the presence of tetracycline.

Compare promoter systems

Components of lacUV

1. lac promoter (lacUV varient)

2. LacI repressor

3. Isopropyl B-d-1-thiogalactopyranoside (IPTG) is an inducer

Default state if OFF. When you add IPTG it turns on. Very useful when you want inducible expression within bacteria

Tet system

1. Tet-Off system: tetracycline prevents the tTA transcription factor from binding at the promoter. Gene expression is inhibited in the presence of tetracycline

2. Tet-On system: Tetracycline binds the rtTA transcription factor and allows it to bind DNA at the promoter. Gene expression is induced in the presence of tetracycline.

T7 expression system

1. T7 promoter, T7 RNA polymerase, Very strong transcription

Very useful when you want very high protein production in bacteria

Name 4 methods of getting vectors into mammalian cells

→Lipid-mediated transfection

→Calcium phosphate

→Electroporation

→Viral transduction

Compare/Constrast transient transfections and stable transfections

Transient Transfection

1. Transfected DNA not integrated into genome, but remains in the nucleus

2. Transfected genetic material is not passed onto the progeny; genetic alteration is not permanent

3. Does not require selection

4. Both DNA vectors and RNA can be used for transient transfection

5. high copy number of transfected genetic material results in high levels of protein expression

6. Cells are typically harvested within 24-96 hrs of transfection

7. Generally not suitable using vectors with inducible promoters

Stable transfection

1. Transfected DNA integrates into the genome

2. Transfected genetic material is carried stably from generation to generation; genetic alteration is permanent

3. Requires selective screening for the isolation of stable transfectants

4. Only DNA vectors can be used for stable transfection; RNA by itself cannot by stably introduced into cells

5. Single or low copy number of stably integrated DNA results in lower level of protein expression

6. Requires 2-3 weeks of selection for the isolation of stably transfected colonies

7. Suitable for studies using vectors with inducible promoters

Define transfection and transduction relative to mammalian expression vectors

Transfection: Introduction of vectors, without using viruses, into mammalian cells

Transduction: Introduction of vectors into mammalian cells using viral-mediated delivery

Design a basic mammalian expression strategy

pee pee poo poo

Define a DNA library and explain its purpose in molecular genetics research

Problem: A genome contains thousands of genes, but cloning usually isolates one fragment at a time

Solution: Create a DNA library- a collection of cloned DNA fragments that together represent an entire genome or transcriptome

Key Idea: A DNA library is a stored representation of genetic information

When is it used?: Gene discovery, genome sequencing, functional genomics, protein expression

Distinguish between genomic DNA libraries and cDNA libraries based on the source of DNA and the types of sequences they contain

Genomic DNA library→ Contain fragments representing the entire genome of an organism. It includes both coding and noncoding DNA

cDNA library→ Collection of DNA fragments made from mRNA molecules. Represents genes that are being actively expression

Outline the major steps involved in constructing a genomic DNA library

1. Isolate genomic DNA

2. Fragment DNA (restriction enzymes or mechanical shearing)

3. Insert fragments into vectors

4. Transform host cells

5. Store cells

Describe how cDNA libraries are generated from mRNA and explain the role of reverse transcriptase in this process

→mRNA isolation

→Reverse transcription

→Creates cDNA: complementary DNA synthesized from RNA templates

Overall Steps

1. Isolate mRNA

2. Use reverse transcriptase to synthesize cDNA from mRNA

3. Covert to double-stranded DNA

4. insert into cloning vector

5. Transform host cells

RESULT: Library representing expressing genes in a specific cell type or condition

Compare advantages and limitations of genomic and cDNA libraries for studying gene structure and gene expression

Advantages:

1. Contain only expressed genes

2. Lack introns and regulatory regions

3. useful for protein expression in bacteria

4. Applications

   1. Identifying expressed genes

   2. Studying gene expression patterns

   3. producing recombinant proteins

Limitations

1. Only genes expressed in sampled cells appear

2. Rare transcripts may be underrepresented

3. No information about

   1. Promoters

   2. Introns

   3. regulatory regions

Identify common cloning vector used for DNA library construction and explain how insert size influences genome coverage

Coverage depends on:

1. Genome size

2. Insert size

3. Number of clones

Higher coverage increases the probability that any gene is represented

Large-insert vectors dramatically reduce clone numbers

1. Plasmids: ~5-10 kb

2. Lambda phage: ~20 kb

3. Cosmids: ~40 kb

4. BACs: ~100-300 kb

5. YACs: up to ~1 Mb

Explain at least two methods used to screen DNA libraries to identify clones containing a gene of interest

→Once library exists, we need to identify the clone of interest

Common screening methods

1. Hybridization screening

2. PCR screening

3. Antibody screening

4. Functional screening

Overall goal is to find one clone among thousands or millions

Examples One: Hybridization sequencing

→ Uses a DNA probe complementary to the gene of interest

1. Transfer colonies to membrane

2. Lyse cells

3. Hybridize labeled probe

4. detect signal

5. positive colonies contain target sequence

Example Two: PCR screening

→Used to scan complex libraries (genomic or cDNA) to isolate specific clones by producing visible DNA bands, which reduces false positives compared to hybridization

1. Pooling: Library to broken down into structured, hierarchical pool to minimize the number of reactions

2. Amplification: Primers designed to the target gene are added to the pools to identify positive locations, as described in this article from Intact genomics

3. Identification: Positive, smaller sub-pool are screened until the specific clone is isolated

Describe the purpose of expression libraries and how they differ from other types of DNA libraries

→Libraries designed so inserted DNA produced protein

Features:

1. Strong promoter

2. Ribosome binding site

3. Translation signals

Purpose: Screen clones based on protein activity or antibody binding

Define terms: dideoxy (chain termination) sequencing

Determines the order of bases by selectively terminating DNA synthesis using modified dideoxynucleotides (ddNTPS).ddNTPS stop strand elongation due to missing 3’-OH group, generating fragments of varying lengths to resolve the seuqnece

Describe the chain termination method for sequencing DNA and how it differs from normal cellular DNA replication

Chain termination: determines DNA sequence by intentionally halting DNA synthesis using modified nucleotides (ddNTPS).

Normal DNA replication contains only dNTPS and terminates when replication finihes, ends of linear chromosomes or colision. Also creates a full length copy of the template strand

Differentiate dideoxynucleotides versus regular deoxynucleotides

→dNTPs are normal bases while ddNTPS are fluorescently labeled

→These fluorescent ddNTPS can not be added onto the next, their 3’ hydroxyl is unavailable

→Each ddNTP has a unique fluorophore

Describe the advantage of sequenase over klenow polymerase

Sequenase: genetically modified T7 DNA polymerase. It has high processivity and efficient incorporation of dideoxy and deoxynucleotides. It also produces more uniform band intensities and has fewer false signals

Describe how DNA fragments separated and detected after chain termination sequencing

Each fragment is typically ran through a gel where it is separated based on size. Laser exites the dye-labeled terminal ddNTPS as they pass a detector, creating a chromatogram that shows the nucleotide sequence/

Describe how a dideoxynucleotide terminates a growing DNA chain

Dideoxynucleotide lacks a 3’-OH group, making it impossible for continue replication

Adapters

Short, synthetic DNA oligos ligated to the ends of DNA fragments to enable amplification on the flow cell, binding to the sequencer, and sequencing primer annealing

Next gen sequencing (NGS)

Set of modern DNA sequencing technologies that perform massive parallel sequencing, allowing for rapid and cost-effective sequencing of large genomes or multiple samples simultaneously

tagmentation

A streamlined library preparation method where a Tn5 transposase enzyme simultaneously cuts (fragments) and tags (ligates adapater sequences to) DNA in one step, replacing traditional multi-step fragmentation and ligation

Barcode sequence

A unique, short DNA sequence added to samples during library preparation that allows multiple samples to be pooled and sequenced together, with the barcode enabling the identification and sorting of reads back to their original sample

Consensus sequence

A representative sequence derived by comparing multiple overlapping sequencing reads (aligned sequences) of the same region, calculating the most frequent nucleotide at each position to resolve errors

Contig

A continuous, assembled seqeunce of DNA formed by aligning and joining together, overlapping sequencing reads, often representing a longer part of a chromosome

Read depth

The average number of times a specific nucleotide position is sequenced, often expressed as 30X or 100X. Higher depth increases accuracy

Emulsion PCR

A method used to amplify clonal, individual DNa templates in separate, water-in-oil micelle droplets. Used commonly in earlier NGS platforms to create polonies

Third gen sequencing

Newer, “longer-read” sequencing technologies, such as Pacific Bioscience and ONT, that sequence single molecules of DNA in real time without needing prior PCR amplification, allowing for much longer reads (thousands to millions of base pairs)

Describe 2 methods of fragmenting genomic DNA or NGS library preparation

Fragmentation by ultrasonic disruption: uses a focused high-frequency acoustic energy to shear DNA within a closed, airtight tube.

Fragmentation by tagmentation:Uses a cocktail of transposases to cleave DNA. Highly efficient and scalable, allowing fragmentation, end repair, and A-tailing to be done in a single tube. It is beneficial for low-input samples, although it may introduce some sequence bias

Describe two methods of creating a target sequencing library for next generation sequencing

PCR: PCR amplify the region of interest then add adaptors to sequence.

Biotinylated oligonucleotide probes: Fragmented DNA is added to biotinylated probes, creating a panel. The probes have sequences complimentary to specific genes or exons

Illumina sequencing

Breaks DNA into fragments, attaching them to a flow cell, and using fluorescently labeled nucleotides to identify bases, allowing millions of sequences to be read simultaneously

→ uses SBS, which tracks the addition of fluorescently labeled nucleotides as the DNA chain is copied, enabling accurate, large-scale, and rapid sequencing

1. Library preparation: DNA is fragmented and adaptors are added to end

2. Cluster generation: fragments are attached to a flow cell (glass slide) and amplified (bridge amplifcation) into distinct, clonal clusters to boost the signal

3. Sequencing: Fluorescently labeled nucleotides are incorporated; a camera records the emission, identifying the base

4. Data analysis: Billions of reads are aligned to a reference genome ,or assembled, to identify variants

5. it requires fluorescence, it is slightly slower than ion torrent, its very accurate and you need an optical imaging system

Ion Torrent sequencing

1. DNA isolation, fragmentation, adding adaptors

2. Partition each fragment into its own location using microbeads

3. Emulsion PCR: Separate the beads using oil and water to avoid contamination. Replicate DNA using PCR, so there are thousands of copies of each DNA fragment

4. Sequencing: Oil is removed and beads are separated into microwells. Primer and polymerase are added. dNTPS are added one at a time and if a dNTP is added, the addition is recorded

5. detects a pH change, it is faster, lower in repeats and requires a semiconductor chip

It is faster than illumina, but it can not differentiate multiple of the same dNTPS in a row

Nanopore sequencing

1. DNA/RNA passes through nanopore protein

2. Changes in ionic current are measured

3. Signal translated into nucleotide sequence

4. Developed my oxford nanopore

5. how does it work?

   1. Motor protein unwinds DNA

   2. Single strand passes through pore

   3. Each base alters electrical current

   4. Machine learning decodes signal

SMRT sequencing

1. High-molecular weight DNA is fragmented and hairpin adapters are ligated to both ends, forming a circular, single-stranded template called SMRTbell

2. The SMRTbell template is loaded into a SMRT cells, which contains millions of nanowells. DNA polymerase is anchored to the bottom of each well and binds to the template

3. As the polymerase incorporates fluorescently labeled nucleotides, each base emits a specific light pulse. This fluorescent label is cleaved off after incorporation, allowing the natural DNA synthesis to continue

4. The polymerase reads the circularized template multiple times in a rolling loop fashion. This multiple passes are analyzed to correct errors

Understand the principles of 3rd generation sequencing

Massively parallel method of sequencing

1. Genomic DNA (gDNA) isolation

2. NGS library construction

   1. gDNA fragmentation

   2. preparing fragments to interact with sequencing platform

3. Library partitioning

   1. Separating fragments of DNA into clusters of identical copies

4. Cluster sequencing

5. Data analysis

Describe nanopore and pacbio technologies

Nanopore: measures changes in electrical current as single-stranded DNA/RNA molecules pass through a protein nanopore in a membrane. Each base causes a characeristic disruption in the current, translated into sequence via machine learning

SMRT: Single-molecule-real-time sequencing uses sequencing by synthesis. A DNA polymerase is anchored at the bottom of a nanopore plate. As it incorporated fluorescently labeled nucleotides, it emits light flashes detected in real time.

Compare Sanger/NGS/TGS methods

1. Sanger: low throughput, high accuracy, short reads

2. NGS(Illumina): High throughput, short reads

3. 3rd Gen: long reads, real-time, single molecule

Application/advantages/limitations of Illumina

Advantages:

1. high accuracy

2. massive throughput

3. Cost effective

4. flexible

Limitations

1. Short read lengths

2. computational burden

3. Initial capital investment

4. Overclustering risks

5. No-real time data

Application/advantages/limitations of Ion Torrent sequencing

Advantages:

1. Rapid turnaround times

2. Lower captial equitment and operational costs compared to others NGS technologies

3. Simplicity and compactness

4. Targeted amplification

5. Scalability

Limitations

1. Homopolymer errors

2. Shorter read lengths

3. lower throughput

4. single-end reads

5. amplification bias

Application/advantages/limitations of Nanopore sequencing

Advantages

1. Ulta long reasons

2. Portable devices

3. real time sequencing

4. direct RNA sequencing

5. lost capital cost

Disadvantages

1. Higher error rates

2. Signal noise

3. Data analysis complexity

4. lower throughput than illumina

Application/advantages/limitations of SMRT sequencing

Advantages:

1. Long reads

2. High consensus accurancy (hiFi reads)

3. No amplifcation bias

4. Detects epigenetic modifications

Disadvantages

1. Higher cost than Nanopore

2. Lower throughput than illumina

3. Specialized equipment required

Differentiate Klenow polymerase and Taq polymerase

Klenow: lower optimal temp (37C), does proof read, high fidelity

Taq polymerase: high optimal temp (70-80C), low fidelity

Describe the following methods: Southern blotting and genomic library construction with both bacteria and phage

Southern blot→ to detect DNA samples

Bacterial libraries

1. Vector are plasmids

2. Genomic DNA and plasmids are cut with the same restriction enzymes. the DNA fragments are ligated into the plmids

3. Transformation: recombinant plasmids are introduced into E.coli cells via transformation

4. Baceria are grown on selective media to identify colonies containing the recombinant plasmid

5. Suitable for smaller fragments

Bacteriophage libraries

1. Lambda phage virus

2. Genomic DNA is partially digested and ligated into the phage genome, replacnig non-essential genes

3. Recombinant DNA is packaged into infectious phage particles in vitro