Bioinformatics 3 Exam

What is PCR

Polymerase chain reaction, a process that allows us to amplify specific pieces of DNA, essential for DNA analysis in molecular biology

What is a GMO?

Genetically modified organisms: An organism of microorganism whose genetic material has been altered by means of genetic engineering

What isis genetic engineering?

Also called genetic modification, is the direct manipulation of organism as genome using biotechnology

How does PCR work?

1)Use primers specific to DNA/gene you are interested in

2) Strands are separated by heat

3) DNA Polymerase bind to double stranded primer and DNA to build a complementary strand

4) Heating and cooling continues to get an exponential increase in copies

(Denature, annealing, extension)

What is the specifics of the process?

1) Denaturation: Heating the DNA to 90-95 C to separate the strands

2) Annealing: Cooling the DNA to 55-60 C to allow primers to bind to the target sequence

3) Extention: DNA polymerase extends the primers, synthesizing new DNA strands at 72 C

How many cycles does a typical PCR process take?

30 - 35 cycles to achieve exponential amplification ( Each cycle double the amount of target DNA)

WHat are some applications of PCR?

1) Genotyping - identifying genetic variations between species and individuals

2) Sequencing - Determining the nucleotide sequence of DNA

3) Visualization - Using agarose gel electrophoresis to visualize amplified DNA

Primers should have a _______ to ensure proper binding and stability.

Balanced GC

What is the result of PCR?

Exponential amplification of the target DNA sequence producing millions of copies.

what is the enzyme used in PCR?

Taq polymerase, is derived from thermophilic bacteria that live in hot springs and can withstand the high temps used in PCR

how long are the primers and what are they designed for? (in PCR)

Primers are typically 18-24 nucleotides long and are designed to bind specifically to the target DNA sequence to initiate amplification during PCR.

What is Electrophoresis?

a technique use to separate DNA fragments based on their size by applying an electric field

What is the process of electrophoresis?

1) DNA samples are mixed with loading dye and placed in wells at one end of the gel matrix (usually agarose)

2) An electric current is applied, causing negatively charged DNA fragments to migrate toward the positive electrode

3) Smaller DNA fragments move faster through the gel matrix, while larger fragments move slower, resulting in separation by size

4) DNA fragments are stained with a dye (red or green) that fluoresces under UV light, allowing visualization of the separated fragments

what are the uses of electrophoresis?

Determining the size of DNA fragments, identifying genetic variations, checking the purity of DNA samples

What are some advantages of Electrophoresis?

easy to perform with basic lab equipment, relatively inexpensive, good for separating fragments of different sizes

what are some limitations of electrophoresis?

limited to separating fragments with significant size differences, provides qualitative rather than quantitative data

what is Sanger sequencing?

AKA: Dideoxy method, used to determine the nucleotide sequence of DNA by incorporating ddNTPs that terminate DNA synthesis

what is the process of sanger sequencing?

1) DNA amplified using PCR to produce many copies of the target sequence

2) DNA is heated to separate the strand

3) A primer binds to the single-stranded DNA

4) DNA polymerase synthesizes new DNA strands, incorporating both regular nucleotides and fluorescently labeled ddNTPs

5) Incorporation of a ddNTP stop DNA synthesis at specific points

6) The resulting fragments are separated by size using capillary electrophoresis

7) A laser excites the fluorescent labels, and the emitted light is detected to determine the sequence

What are the uses of sanger sequencing?

determining the exact nucleotide sequence of DNA, identifying specific genetic variants, detecting mutations in genes

what are the advantages of sanger sequencing?

High accuracy in determining nucleotide sequences, single-base resolution, can be used for various applications, including small-scale sequencing projects

What are the limitations of sanger sequencing?

more expensive than basic electrophoresis, lower throughput compared to next-generation sequencing technologies, limited to relatively short sequences (up to ~ 1000 bp)

what are similarities between electrophoresis and sanger sequencing?

both techniques involve the separation of DNA fragments by size using electrophoresis, both are fundamental techniques in molecular biology and genetics

what are the differences in purpose of electrophoresis and sanger sequencing?

electrophoresis is primarily used for visualizing and separating DNA fragments and sanger sequencing is used to determine the nucleotide sequence of DNA

What is the difference in process complexity between electrophoresis and sanger sequencing

Electrophoresis- more straightforward and faster, involving basic separation and visualization

Sanger sequencing - more complex, involving PCR amplification, incorporation of ddNTPs, and detection of fluorescent label

what is the resolution of electrophoresis and sanger sequencing?

electrophorsis separates fragments with with significant size differences

Sanger Sequencing requires specialized equipment and is more expensive

What is the difference between cost and equipment with electrophoresis and Sanger sequencing

Electrophoresis - requires basic lab equipments and cost effective

Sanger sequencing - requires specialized equipment and is more expensive

What is illumina?

Illumina sequencing uses sequencing by synthesis, where the incorporation of nucleotides into a growing DNA strand is detected in real-time

What is the illumina process?

1) The DNA is fragmented into small pieces

2) Short DNA sequences called adapters are lighted to the ends of the DNA fragments

3) The fragments are immobilized on a flow cell and amplified to form clusters

4) Flourescently labeled nucleotides are added, and each incorporation is detected optically

What are the advantages of illumina?

Illumina sequencing can produce millions to billions of reads in a single run, provides high accuracy and reliable data, relatively cost-effective for large-scale sequencing projects

What is illumina used for?

Used for sequncing entire genomes, analyzing gene expression by sequencing RNA, studying DNA modifications and interations

What length of reads do illumina produce?

Typically produces short reads, around 100-300 bps

Illumina produces ______ of data.

Large amounts

What is a ion semiconductor?

A method that detects DNA sequences by measuring changes in electrical charge rather then using optical methods

What is the ion semiconductor process

1) DNA is fragmented into smaller pieces

2) Adapters are added to the DNA fragments

3) DNA fragments are attached to micro beads

4) emulsion PCR amplifies DNA fragments on the beads

5) The beads are placed in wells on a semiconductor chip

6) Nucleotide are flowed over the chip one type at a i e (A,T,G,C)

7) As nucleotides are incorporated into the DNA strand, hydrogen ions are released, causing a change in pH

8) the semiconductor chip detects these changes in electrical charge, which are used to determine the DNA sequence

What are the advantages of ion semiconductors?

Less expensive than optical sequncing methods, suitable for smaller-scale sequencing projects, can provide rapid sequencing results

What are ion semiconductor challenges?

Difficulty in accurately sequencing regions with long repeats of the same nucleotide (AAAA)

Ion semiconductors focus on _____ rather than _______

Electrical charges/ optical signals

What is Nanopre sequencing?

A third party sequencing technology that allows for the sequencing of long DNA strands by measuring changes in electrical current as DNA molecules pass through nano pores

What is the nano pores process?

1) Single DNA molecules pass through nonopores embedded in a membrane

2) As the DNA passes through the nano pores, it disrupts the electrical current flowing through the pore

3) The changes in current are measured and analyzed to identify the sequence of nucleotides in the DNA strand

What are the advantages of Nanopore sequencing?

Capable of producing ultra-long reads, up to hundreds of thousands of nucleotides

Provides real-time data as the DNA passes through the nanopore

These devices are often portable and can be used in various settings including fieldwork

What are Nanopore sequencing challenges

Higher error rates compared to some other sequencing technologies, through improvements are continually being made

Requires sophisticated algorithms to accurately interpret the changes in electrical current and determine the DNA sequence

What is PacBio?

Pacific biosciences, a third generation sequencing technology that allows for the sequencing of long DNA strands by measuring the incorporation of nucleotides in real time

What is the process PacBio?

1) DNA is prepared by adding adapters to create circular DNA molecules

2) DNA molecules are placed in tiny walls called Zero Mode Waveguides on a SMRT cell

3) As DNA polymerase incorporates nucleotides into the DNA strand, the nucleotides edit light

4) The emitted light is detected and measured to determine the sequence of nucleotides

What are the advantages of PacBio?

Capable of producing long reads, up to tens of thousands of nucleotides, Offers high accuracy through circular consensus sequencing, where the same molecule is sequenced multiple times, can be used for various applications, including genome assembly, transcriptomics, and epigenetics

What are challenges that PacBio faces?

Higher error rates compared to some other sequencing technologies, though circular consensus sequencing helps mitigate this, generally more expensive than second-generation sequencing methods

What role do dideoxy nucleotides (ddNTPs) play? How do researchers determine the sequence with these?

Structure: ddNTPs are modified nucleotides that lake 3’ hydroxyl group (-OH) found in regular deoynucleotides (dNTPs)

Function: The absence of the 3’Hydroxl group prevents the proration of phosodieseter bond with the next nucleotide, effectively terminating DNA synthesis when a ddNTP is incorporated stoping the elongation of the DNA fragment, multiple lengths

Amplify target DNA, separate strands and bind primer, incorporate dNTPs ad ddNTPs to create fragments, separate fragments by size, detect and record fluorescent signals, analyze eletropherogram to determine the sequnce

What’s is sequencing by synthesis?

A methods used in next-generation sequncing technologies to determine the sequence of DNA by synthesizing a new strand and detecting the incorporation of nucleotides in real-time. (illumina and ion semiconductor)

Optical vs Charge

In illumina sequencing, fluorescetly labeled nucleotides emit light when in corporatd, and emitted light os detected by a camera

In ion semiconductor sequncing, the incorporation of nucleotides releases hydrogen ions, causing a change in electrical charge, which is detected by semiconductor chip

Which technologies fragments genome before sequncing?

Illumina and ion semiconductor

Approximately how long are the reads that are produced by each technology? (Short vs. Long)

Illumina - 100 - 300 bp

Ion semiconductor - 100 -400 bp

PacBio - produce very long reads, up to ten of thousands oof base pairs

Nanopore Sequning - hundreds of thousands of base pairs

What are the Robles that can arise when assembling a genome?

Uneven coverage, Random fragmentation, repetitive DNA, tandem repeats, Homopolymer Regions, Structural variations, GC - Rich or AT -rich regions, Sequencing errors, error correction, contig assemly, scaffolding

What are reads?

Short sequences of dan that are generated by sequencing technologies. They represent fragments of original DNA same and they are used to reconstruct the genome during the assembly

What are paired-end and single end reads and how do they differ?

Single - end reads are sequecnes obtained from one end of a dna fragments. Only one end of the fragment is sequenced, providing a signal read per fregment

Paired-End reads - are sequences obtained for both ends of DNA fragments. Both ends of the fragments are sequenced providing two reads per fragment

Single end reads - provide information from one end of the fragment, offering less context about the overall structure of the genome

Paired end reads - provide information from both ends of the fragments, offering more context and helping to resolve complex regions

What are contiguous and scaffolds? How do they differ?

• Contigs (short for contiguous sequences) are continuous sequences of DNA that are assembled from overlapping reads. They represent segments of the genome where the sequence is known without gaps.

• Scaffolds are collections of contigs that are linked together using additional information, such as paired-end reads or long reads, to span gaps between contigs. Scaffolds provide a larger, more comprehensive assembly of the genome.

• Contigs: Continuous sequences without gaps.

• Scaffolds: Collections of contigs linked together, may contain gaps.

• Contigs: Provide known sequences within uninterrupted segments.

• Scaffolds: Provide a larger assembly with information about the relative positions of contigs, including gaps.

Assembly Process:

• Contigs: Formed by aligning and merging overlapping reads.

• Scaffolds: Formed by linking contigs using paired-end reads, long reads, or other sequencing data.

Usage:

• Contigs: Used to represent known sequences within segments of the genome.

• Scaffolds: Used to represent a more comprehensive assembly of the genome, including the relative order and orientation of contigs.

describe the difference between de novo and referneced based assembles

• De Novo Assembly: Uses only the sequencing reads obtained from the target genome.

• Reference-Based Assembly: Uses sequencing reads and an existing reference genome.

Computational Requirements:

• De Novo Assembly: Requires more computational resources and complex algorithms.

• Reference-Based Assembly: Requires fewer computational resources and simpler algorithms.

Accuracy:

• De Novo Assembly: Can be less accurate in repetitive or complex regions.

• Reference-Based Assembly: Provides higher accuracy due to the guidance of the reference genome.

Applicability:

• De Novo Assembly: Suitable for organisms with no existing reference genome.

• Reference-Based Assembly: Suitable for organisms with an existing reference genome.

What are the principles of gene expression analysis?

• Gene expression analysis involves measuring the amount of mRNA in a sample to determine which genes are active and to what extent.

• Different technologies infer the starting amount of mRNA by converting it into a measurable signal, such as fluorescence, radioactivity, or sequencing reads.

Explain the differences between the different gene expression analysis methods.
Northern Blots:

• How They Work: RNA samples are separated by gel electrophoresis, transferred to a membrane, and hybridized with a labeled complementary probe.

• Requirements: Known sequence information to design the probe.

• Target: Measures one target gene at a time.

• Measurement: The intensity of the signal (brightness or darkness) on the blot indicates the amount of RNA bound by the probe.

qPCR (Quantitative PCR):

• How It Works: RNA is reverse transcribed into cDNA, which is then amplified using PCR with fluorescent dyes that bind to double-stranded DNA.

• Requirements: Known sequence information to design specific primers.

• Target: Measures one target gene at a time.

• Measurement: The fluorescence intensity correlates with the amount of cDNA, which reflects the starting amount of mRNA.

Microarrays

• How They Work: RNA samples are labeled with fluorescent dyes and hybridized to a microarray chip containing thousands of oligonucleotide probes.

• Requirements: Known sequence information to design the probes on the array.

• Target: Measures many genes simultaneously.

• Measurement: The fluorescence intensity at each spot on the array indicates the amount of RNA hybridized to that probe.

RNAseq (Transcriptomics):

• How It Works: RNA is reverse transcribed into cDNA, which is then sequenced using high-throughput sequencing technologies.

• Requirements: Does not require known sequence information, but having a reference genome helps with alignment.

• Target: Measures many genes simultaneously.

• Measurement: The number of sequencing reads aligned to each gene reflects the amount of mRNA in the sample.

You should be able to interpret the results of these different techniques, like
what we walked through in class. You should also be able to draw what the
results would look like for some of these.

• Interpretation: The intensity of the bands on the blot indicates the relative amount of RNA. Darker bands represent higher expression levels.

qPCR:

• Interpretation: The cycle threshold (Ct) value indicates the number of cycles needed to reach a detectable level of fluorescence. Lower Ct values indicate higher initial amounts of mRNA.

Microarrays:

• Interpretation: The color and intensity of each spot on the array indicate the relative expression levels of the corresponding gene. Red spots indicate higher expression in the experimental sample, green spots indicate higher expression in the control, and yellow spots indicate equal expression.

RNAseq:

• Interpretation: The number of reads aligned to each gene indicates the relative expression levels. Higher read counts correspond to higher expression levels.

Drawing Results

Northern Blot:

• Example: A gel with bands of varying intensity. Darker bands indicate higher expression.

qPCR:

• Example: A plot of fluorescence intensity vs. cycle number. The point where the curve crosses the threshold indicates the Ct value.

Microarrays:

• Example: A grid of spots with varying colors and intensities. Red, green, and yellow spots indicate different expression levels.

RNAseq:

• Example: A bar graph showing the number of reads aligned to each gene. Higher bars indicate higher expression levels.

How have humans been altering the DNA of organisms over the last several
thousand years?

• Selective Breeding: For thousands of years, humans have been selectively breeding plants and animals to enhance desirable traits

  . This involves choosing individuals with beneficial characteristics and breeding them to produce offspring with those traits.

• Crossbreeding: Combining different varieties or species to produce hybrids with desired traits

Modern Genetic Engineering:

• Recombinant DNA Technology: In the 20th century, scientists began using recombinant DNA technology to directly modify the genetic material of organisms

  . This involves transferring genes from one organism to another to achieve specific traits.

• CRISPR: More recently, CRISPR technology has allowed for precise editing of genes, enabling targeted modifications

• Domestication of Dogs: Selective breeding of wolves to produce domesticated dogs with traits beneficial to humans

• Cultivation of Crops: Selective breeding of wild plants like teosinte to produce modern corn

• Genetic Engineering: The creation of the Flavr Savr tomato, the first commercially available GMO, engineered for longer shelf life

Be able to describe (at a basic level) the examples I walked through in class.

• Brassica oleracea: The wild cabbage species from which various vegetables like cabbage, kale, broccoli, kohlrabi, brussels sprouts, and cauliflower have been derived through selective breeding

• Brussels Sprouts: Modern varieties have been bred to reduce bitterness, making them more palatable

What are some traits that have been the targets of genetic modification? Give
examples.

1. Herbicide Tolerance:

   • Example: Roundup Ready crops that are resistant to glyphosate, allowing farmers to use herbicides without damaging the crop.

2. Insect Resistance:

   • Example: BT crops that produce a protein from Bacillus thuringiensis, which is toxic to specific insect pests but harmless to humans.

3. Improved Nutrition:

   • Example: Golden Rice engineered to produce beta-carotene, a precursor to vitamin A, to combat vitamin A deficiency

4. Disease Resistance:

   • Example: Papayas engineered to be resistant to the ring spot virus, saving the Hawaiian papaya industry

5. Stress Tolerance:

   • Example: Crops engineered to be more resilient to drought, salinity, and other environmental stresses

6. Storage and Shelf Life:

   • Example: Arctic apples and non-browning potatoes that resist oxidation and maintain their appearance longer

You should be familiar with the topics and examples covered in the Kurzgesagt

The Kurzgesagt video on GMOs covers the history, benefits, and controversies of genetically modified organisms

. It explains how GMOs are created, their applications in agriculture and medicine, and addresses common concerns about their safety and environmental impact