Genetics module 11

Define Type I restriction endonucleases

Recognize specific DNA sequences and cleave the DNA somewhere else. They restrict the entry of foreign DNA into the bacterial cells. Not very useful in molecular biology

Define Type II restriction endonucleases

Cleave within the recognition site. Restriction sites are palindromes. Cutting creates “sticky” ends

Why don’t bacterial restriction endonucleases attack the host’s own DNA?

The host commonly methylates a base in every copy of the RE site in its own genome.

What happens to DNA sequences cut be Type II REs?

They can be rejoined by ligases. It helps connect DNA sections.

How does gel electrophoresis sort DNA/RNA fragments?

They are sorted by size. Negatively charged DNA bases move towards the positive electrode. Must occur in a gel.

How is an agarose gel prepared?

• Acquire gel tray

• Prepare barriers to retain agarose

• Pour molten agarose in trays

• Insert comb to form wells before agarose solidifies

• Load DNA samples in wells and apply voltage

How is size-fractionated DNA visualized?

Via a DNA binding fluorescent dye. Ethidium bromide (EtBr) is an intercalating dye that could cause mutations. A camera can visualize the fluorescence.

What is the relation between migration rate of linear DNA and the log of its molecular mass / number of base pairs?

The less base pairs, the further it travels in a gel

Other than molecular mass / base pairs, what else can affect the mobility of DNA fragments in a gel?

• Agarose concentration in gel

• DNA topology

• Voltage

How does agarose concentration in a gel affect DNA migration?

Increased agarose concentration = decreased pore size. Smaller pores are more resistant to DNA movement, and favor small fragments, so it’s best for visualizing differences in small size DNA

What are the topologies of DNA and how do they affect migration in gel?

• Linear: Travel moderately well

• Relaxed circular: Travels the least, takes up the most space

• Supercoiled: often the conformation found in cells. Can be circular or linear. Travels furthest.

What are some factors that do not affect DNA molecule migration in agarose gel?

%GC of the molecule or the sequence of the molecule has no effect

What are the minimum requirements for DNA synthesis in vitro?

• A strand of DNA to act as a template

• A short, single strand of DNA (primer)

• DNA polymerase

• dNTPs

• Mg+ (for the polymerase)

What was Mullis’ insight on PCR?

It is an enzymatic copying of dsDNA using 2 primers, complementary to the opposite strands, which would lead to exponential increase in amount of target sequence.

What are the steps and corresponding temperatures for PCR?

• Denature: 95

• Anneal: 60

• Extend: 75

How many PCR cycles are generally performed in an experiment?

30-35. Theoretically allows for more than a billion fold amplification of target DNA, assuming that the reaction occurs with 100% efficiency

Discuss what happens in denaturation

Temperature is about 95 degrees. dsDNA melts into ssDNA.

Discuss what happens in annealing

Temperature 65. Primers are able to bind to their complementary sequences. Temperature can vary based on the melting point (Tm) of the DNA sequence and the salt concentration (since it affects the melting point)

Discuss what happens in elongation

Temperature is about 75. Polymerase binds the annealed primers and extends DNA 5’-3’

What are the necessary ingredients in PCR?

• dNTPs

• Magnesium (for polymerase)

• Primers (usually 2)

• Template DNA (ssDNA or dsDNA)

• Thermostable DNA polymerase (usually Taq)

• Salts

• pH control

• Stabilizers

Explain the importance of PCR primers and what they do

They are short oligos of ssDNA that anneal to opposite strands, so they can help create an exponential growth product. The size of the PCR product depends on how far apart the annealing sites are. Most PCR is 2 kb or less but can be up to 40 kb.

Why are primers usually 18-25 bp long?

Allows for increased specificity; small primers may not be specific enough, long primers don’t really increase specificity and are more expensive. 18-25 is usually enough to match only the intended sequence.

What are some applications of PCR?

• Amplifying target genes for study

• Detection of rare sequences using desired primers

• Detection of contaminants

• Detection of different bacterial, pathogenic, etc.

• Detection of DNA in crime scenes

• Environmental DNA (eDNA)

What is the threshold in PCR?

A predetermined number that after which DNA is amplified exponentially

Explain why the growth of DNA in PCR is exponential

In early stages, growth is limited by the amount in the previous cycle. It then gains enough to grow exponentially. When polymerase is saturated growth is linear. In later cycles, dNTPs are less abundant. Polymerase may start to wear out, dNTPs run out and growth plateaus.

How is DNA quantified in each cycle of qPCR?

• Using a reporter dye

• Often SYBR green, which fluoresces strongly when bound to dsDNA

• Binds to minor groove in dsDNA

• Other methods: fluorescent probes that can track multiple reactions with multiple targets.

What are some applications of qPCR?

• Quantify the amount of starting DNA of a particular sequence

• Measure the rate at which a gene is transcribed (requires conversion of mRNA to cDNA using RT)

What is the log-linear phase of DNA growth in PCR?

When in a logarithmic scale, DNA growth in the exponential phase appears linear. It is the best way to estimate the amount if starting DNA

What is Cp?

The detection threshold of DNA in qPCR

Describe the Sanger-dideoxy chain terminating method.

Uses modified dNTPs known as ddNTPs that terminate the synthesis. Synthesizes multiple chains of various lengths thanks to terminating nucleotides, which can then be ordered to find out the sequence. Different fluorescence pieces are attached to each kind of ddNTP.

How is fluorescent sequencing detected in Sanger dideoxy sequencing?

Generally automated. We use gel electrophoresis to separate the ssDNA by size and then sequence it through a laser that detects the fluorescence.

What are the pros of Sanger dideoxy sequences?

• Very accurate

• Relatively long sequence reads

• Can be automated

• Low cost (for small samples)

What are the cons of Sanger dideoxy sequencing?

• Too slow for stuff like genome sequencing

• Costly when scaled up

• Requires purification and preparation of each sample

When single sample sequences is too slow, what is the solution?

Massive parallel sequencing. This is the ‘next generation sequencing’ methods (NGS)

How does Illumina DNA sequencing work?

1. Randomly fragment genomic DNA and ligate adapters to both ends of fragments

2. Attach DNA to inside surface of flow cell channel

3. Unlabeled nucleotides and enzymes initiate solid-phase bridge amplification

4. Fragments become double stranded

5. dsDNA denatured to ss

6. Amplification in flow cell channel

7. Determine first base via laser, image base, second base, etc.

8. Align DNA

How do we achieve the short segments of DNA required for Illumina sequencing?

Accomplished via shearing or use of short PCR products

What do adaptor sequences do in Illumina sequencing and what are they used for?

They are added by ligation to DNA ends. They enable attachment to oligonucleotides on the surface of the flow cell

What does “bridge amplification” do in Illumina sequencing?

Used to amplify single DNA molecules into clusters of identical DNA molecules

How does sequencing occur in Illumina DNA sequencing?

By addition of fluorescently labeled nucleotide analogs, 1 base at a time. These analogs are reversible and chain terminators.

What happens after each dNTP is added in Illumina sequencing?

The sequencer pauses and exposes the flow cell to the laser to take a picture of the DNA base incorporated in a cluster.

Explain the basis of Nanopore sequencing

• Not sequencing by synthesis

• Single molecule at a time (no PCR)

• Enzyme unwinds DNA, ss pulled through membrane pore by electrical current

• Each base produces specific disturbance in electrical current used to read the base

What are the pros of Nanopore sequencing?

• Reads up to 100kb

• No amplification required

• Highly portable, connects to USB port

• Can be used in the field

  • Detects methylated bases

What are the disadvantages of Nanopore sequencing?

• Slightly less accurate than other methods

Comparing Sanger, Illumina and Nanopore: Which one(s) are massively parallel?

Illumina and Nanopore

Comparing Sanger, Illumina and Nanopore: Which one(s) sequence via synthesis?

Sanger and Illumina

Comparing Sanger, Illumina and Nanopore: Which one(s) use only a single DNA molecule?

Nanopore

Comparing Sanger, Illumina and Nanopore: Which one(s) use chain terminators, reversible or irreversible?

Sanger (irreversible) and Illumina (reversible)

Comparing Sanger, Illumina and Nanopore: How accurate are they?

Sanger and Illumina up to 99.99%, Nanopore 98-99%

Comparing Sanger, Illumina and Nanopore: What are the reading lengths?

• Sanger: 650-100 bases

• Illumina: 75-600 bases

• Nanopore: >100 kilobases