Cloning

Question 1

What are the two ways in which DNA can be cloned?

Answer 1

1. PCR: easiest, always try firsy
2. Cloning vectors: needed when PCR doesn’t work

Question 2

What are the targets of cloning?

Answer 2

DNA or mRNA.

Question 3

What two types of libraries can be created through cloning?

Answer 3

• cDNA library: mRNA
• Genomic library: DNA

Question 4

What is cloning for?

Answer 4

Genetic cloning involves the amplification of DNA or RNA with the aim of obtaining large quantities of defined pieces of DNA. In order to seek out specific genes, DNA libraries are needed.

Question 5

What is a genomic library?

Answer 5

Genomic libraries are made up from the entire genome of an organism, and are a pool of recombinant molecules such as YACs, bacteriphages, BACs, or plasmids that have nucleic acid inserts (usually overlapping) that cover the entire genome.

Question 6

How can genes be mapped using libraries?

Answer 6

• To map genes the suspected locus needs to be found in the library (screening). This can be done using known sequences as probes, which are associated sequences or from other species.
• Clones carrying the region of interest are identified, and may be overlapping in the DNA they contain.
• DNA can be cut from these vectors and placed into smaller vectors for manipulation, e.g. starting with a YAC and placing the DNA into plasmids in order to sequence the DNA.
• There are several YAC libraries covering the human genome for this purpose. The smaller vectors allow sequencing of the DNA inserts, which can be lined up to form the full sequence of the gene and its surrounding DNA.
• Searching genomic databases allow regions to be found that are indicative of gene start codons, promoter regions, splice regions, and exons.

Question 7

Outline the 6 main steps in making a library.

Answer 7

1. Target DNA fragmentation (partial digestion)
2. Vector linearisation
3. Ligation
4. Transformation/transfection
5. Library screening
6. Clone characterisation

Question 8

How is target DNA fragmented? How is it ligated?

Answer 8

Target DNA fragmentation is done mainly, but not exclusively, through the use of restriction endoucleases that display sequence specificity for specific restriction sites. This generates either sticky ends (EcoRI) or blunt ends (BalI). The average length of the DNA fragments are 4ⁿ, with n being the number of nucleotides in the restriction recognition sequence

Ligation of the fragments and linearised vector molecules is done by mixing the two in a tube, causing the sticky ends to anneal to each other. T4 DNA ligase can be used to anneal both sticky and blunt ends, in which is forms a phosphodiester bond between the 3’-OH and 5’-P groups of the sticky ends. This forms recombinant molecules.

Transformation can be done using a variety of techniques, including heat shock and electroporation. Hosts can either be prokaryotic (E. coli) or eukaryotic (S. cerevisiae), the cells being transformants once they have taken up the recombinant DNA. These form the genomic library.

Question 9

How are DNA fragments linked together? How are libraries probed?

Answer 9

Chromosome walking

Fragments are linked together through chromosome walking, which is where probes detect the desired sequence in the genomic library, and the probes can sometimes overlap with each other: this can be used to piece together the DNA.

Screening the library can be done using either a radioactive probe, or a cDNA probe. After identification of a sequence in a plaque, the DNA can then be sequenced.

Question 10

Outline the 7 main steps in cDNA cloning.

Answer 10

1. Isolation and purification of mRNA
2. Synthesis of cDNA copy
3. Vector linearisation
4. Ligation
5. Transformation/transfection
6. Library screening
7. Clone characterisation

Question 11

What vectors are used for cDNA cloning?

Answer 11

Plasmids and bacteriophage lambda.

Question 12

How is cDNA synthesised?

Answer 12

Synthesis of a cDNA copy is done through a series of experimental steps:

• Affinity chromatography using an oligo-dT matrix (detects the poly-A tails on mRNAs)
• Strand synthesis using reverse transcriptase to make a DNA/RNA hybrid
• RNase H removes the RNA
• dsDNA is formed using polymerase
• S1 nuclease removes hairpin loop
• Manipulation of dsDNA ends is done to facilitate insertion into vectors, e.g. adaptors, linkers

Question 13

Outline PCR.

Answer 13

Polymerase chain reaction (PCR) can be a shortcut to the desired clone, but some prior information of the sequence of the cloned DNA is needed beforehand. The chemical reaction goes as follows:

1. DNA denaturation and strand separation at 94 ºC
2. Primer annealing at 50-65 ºC
3. Primer extension at 72 ºC
4. This cycle is repeated 20-45 times

Amplification of the DNA is exponential, after 20 cycles there is a 10⁶ times amplification. The amplified ideal DNA length is 200-300 bp, but can be up to 10 kb. Primers usually have a built-in sequence to facilitate subsequent In vivo cloning.

Question 14

What makes a good primer?

Answer 14

Primer design:

• Size- too small means it will bind to more than one site on the genome, if it’s too large it will take a long time to hybridise and would slow down the PCR cycle (none above 30 bp).
• Melting temperature (Tm)- the temperature at which the primer will dissociate. The annealing temperature should be about 5 ºC below the Tm, and both primers should have about the same Tm (usually 55-65 ºC) .
• Should have a G or C at the 3’ end.
• Absence of dimerisation capability.
• Absence of significant hairpin formation.
• Low specific binding at the end of the primer to avoid mispriming.

Question 15

What is TA cloning?

Answer 15

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products.

TA cloning is a subcloning technique that avoids the use of restriction enzymes and is easier and quicker than traditional subcloning. The technique relies on the ability of adenine (A) and thymine (T) (complementary basepairs) on different DNA fragments to hybridize and, in the presence of ligase, become ligated together. PCR products are usually amplified using Taq DNA polymerase which preferentially adds an adenine to the 3’ end of the product. Such PCR amplified inserts are cloned into linearized vectors that have complementary 3’ thymine overhangs.

The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase adds an A to the end of the PCR product. Ligate into a special vector with a terminal “T-tail”. Add a restriction site to the end of your primer and clone in the normal way!

Given that there is no need for restriction enzymes other than for generating the linearized vector, the procedure is much simpler and faster than traditional subcloning. There is also no need to add restriction sites when designing primers and thus shorter primers can be used saving time and money. In addition, in instances where there are no viable restriction sites that can be used for traditional cloning, TA cloning is often used as an alternative. The major downside of TA cloning is that directional cloning is not possible, so the gene has a 50% chance of getting cloned in the reverse direction.