BIO 200 - DNA cloning, experimental gene expression, introduction to genes

Using gels to separate DNA: where are the shorter fragments?

At the bottom

Where will the 5’ and 3’ ends be in the gel for sequencing?

5’ at the bottom, 3’ at the top

In illumina sequencing, why do a PCR step first?

To obtain a lot of copies of the DNA to be able to see the fluorescent molecules.

Does single-molecule sequencing require PCR?

No, they don’t need the volume of DNA to work effectively

Where does the PCR step occur in illumina sequencing?

On the surface of the plate (?)

How does nanopore sequencing work?

There is a voltage across the membrane, when the DNA goes through the pore there is a change in the voltage depending on the base size - permitting people to be able to read the DNA sequence. Only one strand goes through the pore. Read one strand, then the other.

What are some advantages to nanopore sequencing?

• can single thousands of base pairs on one DNA molecule.

• Don’t need to figure out how to assemble the sequence in a long DNA molecule.

• Can know the sequence even when it is repetitive

• Sequencer is small and portable - can take it with a computer in the field

What is recombinant DNA?

Combination of DNA from two sources

What is a vector?

DNA that is easily copied by cells. It is often coupled with another type of DNA like chromosomal DNA.

What is the most common vector?

Plasmids

Where are plasmids found?

In bacteria in nature, there is a huge diversity of them

Why is plasmid replication special?

They replicate on their own, with their own origin of replication, they also regulate themselves

How many copies of plasmids per cell?

10-20 copies per cell

What form do plasmids used in the lab take?

Circular dsDNA molecules

What are restriction enzymes?

Enzymes which recognize a specific sequence and cut phosphodiester bonds in a symmetrical fashion

What are sticky ends?

Ends with a little bit of single stranded DNA which can be re-annealed

What are the steps to generating a recombinant DNA molecule?

• Cut the plasmid with restriction endonucleases

• Anneal sticky ends to chromosomal DNA

• Use a ligase (usually T4) to ligate the two ends together

What are some important parts of a plasmid?

• Origin of replication

• Selection marker - gene that codes for antibiotic resistance

• Polylinkers (multi-cloning sites)

What is contained within a polylinker?

Multiple resitriction sites which are recognized by multiple restriction enzymes

Why is it useful to have multiple restriction sites?

Restriction sites often has a sequence of 6 nucleotides, there is a possibility that a DNA molecule contains at least one of the restriction sites. 1/4000 base pairs of a sequence of 6 nt. You don’t want to cut the DNA.

Why isit advantageous to use more than one restriction enzyme?

You can choose how it will be oriented in the DNA

What is a characteristic of restriction sites?

They are palindromes - same forwards and backwards on the other strand

What is the first step for cloning DNA?

Ligate a DNA fragment to be cloned onto the plasmid vector.

What is the second step for cloning DNA?

Mix E.coli with plasmids in the presence of CaCl2 and a heat pulse. Salt helps pores to open when we give the cell heat shock

What is the third step for cloning DNA?

Spread cells on agar plates with an antibiotic. Cells with plasmid will survive due to their selection marker and make colonies on the agar.

How can you amplify DNA from this method?

Take the colonies, amplify them and extract the plasmid.

What is a DNA library and how can it be formed?

Permanent collection of genes. Cutting the genome with restriction enzymes to generate many fragments. You can then clone them into a vector

What do genomic libraries contain?

Chromosomal DNA

What does a cDNA library contain?

mRNA in a given sample

Why is it more useful to use cDNA mRNA for eukaryotic genomic libraries?

Complement to mRNA because those are the coding regions without introns

What is reverse transcrpiptase?

Enzyme which converts RNA into DNA

When the mRNA is processed, what is added as a tail?

Multiple As (3’ poly A tail)

What does the primer (DNA primer) contain?

Only Ts

What kind of nucleotides does the reverse transcriptase add?

DNA nucleotides

How do you get double stranded DNA from this?

Get a new primer of the 3’ end of the DNA (synthetic) and put a complementary primer to start the synthesis of the double strand.

What is the final step to cDNA library formation?

Adding a linker of fragment of DNA at the two ends that contains a restriction enzyme - ligate so that you can put it in a plasmid

What can recombinant DNA be used for?

• Microarray and in situ hybridization to look at mRNA expression, co-regulation, localization

• Recombinant DNA expression vectors - regulated expression of exogenous genes, and production of proteins, in prokaryotes and eukaryotes

What kind of probe is generated for in situ hybridization?

A DNA probe is generated, complementary to mRNA in the cell - short DNA fragment which is labelled like being recognized by an antibody.

What two things need to happen to the cells for in situ hybridization?

The cells get treated chemically to preserve the structure and they also permeabilize the cell so that probe and antibodies can get inside.

What can microarray analysis study?

Levels of many mRNAs

What is the first step in microarray analysis?

Generate the microarrays - slides with droplets that are then covalently linked to the surface. The different droplets contain different coding sequences for different genes.

What is the second step of microarray analysis?

Prepare samples: Two cells in culture, extract mRNA and make cDNA and label one of the cultures as green and the other as red - one with serus as red, one without serum as greeen

What is the third step of microarray analysis?

Hybridize cDNA so that it sticks to the surface of the slide, obtaining different colours of fluorescence

What can you tell based on the colours of fluorescence seen?

If the particular gene was more abundant before or after the serum was added. Ex. If a spot is green, expression of that gene decreases after serum addition

What can cluster analysis identify?

coordinately regulated genes. Can group genes depending on how they react to the experiment

How can recombinant DNA be used to help humans?

Can take proteins from humans, clone their gene into plasmids put them into bacteria and make the bacteria produce human proteins. Ex. Insulin

How can you study the function of genes in humans?

Clone particular cDNA molecule onto a vector that contains a viral origin of replication. So now original replication from a virus that can replicate in human cells.

How can you introduce plasmids into cultured cells?

lipid treatment or electroporation, an electrical shock

Why is it called transient transfection?

After some divisions, the cells will lose the plasmids

How does stable transfection differ from transient transfection?

The origin of replication on the plasmid is not viral, so the vector undergoes homologous recombination with the genome of the cell