LECTURE 6 (cDNA libraries)

Question 1

What is a cDNA library?

Answer 1

A library showing the cDNA of an organism

Question 2

Main difference between genomic and cDNA libraries?

Answer 2

In genomic; it doesn’t matter where you take the DNA from

In cDNA: cDNA works depending on how were the genes in that stage extracted

Question 3

How is a cDNA library created? 6 steps

Answer 3

• extract the RNA
• 1st strand synthesis
• 2nd strand synthesis
• prepare ends
• clone into appropriate vector
• infect bacteriophage

Question 4

Describe the 1st strand synthesis of cDNA libraries.

Answer 4

You have your mRNA fragment with the polyadenilated tail, you add the primer that binds to the tail (oligo dT) and RT will synthesize the strand (recognizes the ds sections) adding dNTP at the end of this stage you have an RNA-DNA hybrid.

Question 5

Describe the 2nd strand synthesis of cDNA libraries.

Answer 5

There are two main ways:

• First of all you denature the mRNA (use RNase A) and get only one strand of ssDNA, at the 3’ end it tends to form a loop that will be extended with DNAP I, then the loop is chopped off by nuclease S1
• dNTPs are added and the hybrid is treated with polymerase and RNAse H which will chop off some pieces of RNA leaving pieces of cDNA that serve as primers so can be extended with DNAP I , T4 ligase is also used to ligate the different bits (this method has a problem which is orientation)

Question 6

What is directional cloning?

Answer 6

This is a system to make sure your insert is gonna be in the right direction. your mix of primers+ your sequence+ dCTPs to make a methylated DNA because the RE that is gonna be used (XhoI is sensitive to methylated ends)

Question 7

How can we get rid of partial RNA and ensure a full length RNA for the synthesis of cDNA for cDNA libraries?

Answer 7

partial RNA won’t have the cap at the 5’ end therefore if we add 5’ polyphosphatase which dephosphorylates the uncapped strands (therefore no possibility of oligo binding). Then we remove the caps with an enzyme and phosphate in these strands will still be there because polyphosphates couldn’t act on them because they had a cap protecting , we add oligos and the synthesis can start. Then this full length RNA can be used as a template for cDNA.

Question 8

What are the main vectors used for cDNA libraries? why?

Answer 8

bacteriophages e.g lambda phage, efficient and easy to screen

Question 9

How are cDNA libraries constructed with phages? (3)

Answer 9

• sub cloning using RE –> normal method used for everything you insert your insert into a plasmid then into phage
• In vivo excision I: using helper phages
• In vivo excision II: using Cre-lox

Question 10

What do we use to ensure that the mRNA strand is valid to synthesize a cDNA strand for a library?

Answer 10

• 5’ end the cap

- 3’ end poly A tail.

Question 11

Why is the poly A tail important for making cDNA libraries? do all types of RNA have the poly-A tail?

Answer 11

Only mRNA has the poly A tail which binds to oligo dT (T residues) therefore in a mix of m/t/rRNA if we add oligos dT only mRNA will anneal and we can remove the rest to ensure we only have mRNA.

Question 12

Is any type of RNA used for a cDNA library?

Answer 12

No, it has to be mRNA

Question 13

Why is RACE PCR a good method of making cDNA libraries?

Answer 13

because it’s Rapid Amplification of cDNA ends, which ensures full length cDNA because sometimes RT doesn’t make it to the 5’ end this can be also solved by using random primers instead of oligo dT because RT recognizes ds sections.

Question 14

What are arrayed cDNA libraries? And ordered libraries?

Answer 14

Collection of clones that are stored individually. You find which clones overlap and can arrange them in order.

Question 15

What is in vivo excision in cDNA libraries?

Answer 15

It’s a way of creating plasmids which contain inserts for creating a cDNA library.

Question 16

Describe in vivo excision I: using helper phage (for cDNA libraries)

Answer 16

We can use lambda phage which contains a plasmid (pBluscript) which is recognized by other phage and this ensures the in vivo excision (occurs in E.coli) of the plasmid to the other phage. You have your lambda phage (λZapII) with the pBluescript and a ExAssist phage. Both infect E.coli.When E.coli is infected three types of phages are produced; λZapII(unable to infect), ExAssist (unable to replicate) and a new phage particle made of the ExAssist coat containing a pBluscript genome. Then you infect again and end up with a bacteria with pBluscript which will replicate as a plasmid (contains the insert of interest)

Question 17

Describe in vivo excision II: using λTriplEx phage.

Answer 17

λTriplEx phage contains pTriplEx phagemid integrated in its genome which is flanked by twol loxP sites. If this phage infects a bact that contains cre recombinase, the two loxP will excise and recombine to form a phagemid and an empty phage genome. The phagemid will then replicate as a plasmid (contains the insert of interest).

Question 18

What is the quality control of cDNA libraries? (5)

Answer 18

• fullness of length
• Representative clones
• minimise chimaeras
• good insert size
• limit the amount of empty vectors

Question 19

What are chimaeras?

Answer 19

molecules ligated that you didn’t want them to be ligated.

Question 20

Apart from screening cDNA libraries like genomic libraries (using probes and PCR) are there other methods?

Answer 20

yes, activity screens and immunoscreening.

Question 21

How are the probes for screening cDNA libraries?

Answer 21

either come from RNA therefore cDNA probes or portions of genomic DNA therefore gDNA probes.

They can be homologous (from the same spp) or heterologous (coming from diff spp)

Question 22

What is back translation?

Answer 22

IT can’t occur in nature. it’s the reverse of normal translation, can be done with a computer we determine the aa sequence of a protein however we have to be very careful with the degeneracy of the genetic code.

Question 23

Describe immunoscreening for cDNA libraries.

Answer 23

It uses antibody to screen libraries. If a protein is expressed you can raise an antibody against it, the antibodies have a fluorescent protein and will bind to the cells that have the protein expressed

Question 24

What are homologous and heterologous probes?

Answer 24

Homologous: same spp
Heterologous: diff spp.

Question 25

Describe activity screens for cDNA libraries.

Answer 25

It’s used to screen complex proteins. You can insert DNA into complexes that can express it and translate it and leads to the production of a protein e.g insert RNA into oocytes and they’ll translate it. You do this until you get the clone that you want.

Question 26

What are “degenerate primers”?

Answer 26

A collection of oligonucleotides with different base composition at certain sites - sometimes called guessmers

Primers that can be used in PCR, designed on the basis of “reverse translation” of a known protein sequence

Question 27

Problems with immunoscreening.

Answer 27

• antibodies aren’t sometimes specific enough and therefore might hybridize with unrelated proteins
• Postranslational modification is needed (expression of the protein is a requirement)
• The recombinant protein is toxic to the organism that carries the library

Question 28

What is a “full-length” cDNA clone?

Answer 28

• A clone that contains the entire open reading frame

- A clone whose insert is as long as the mRNA produced by the corresponding gene