Primers

What is a primer?• Primers are oligonucleotides, small pieces

of RNA or DNA up to 30 base pairs long

(a bigger piece is known as a polynucleotide)

• Primers are essential for DNA replication

• During replication DNA polymerase must have an attachment point (a free 3’-OH) or it won’t work

What is a primer? ctd.

• In natural systems an enzyme, primase, (which doesn’t require the attachment point) synthesizes a short RNA sequence complementary to the template DNA strand

• This binds to the template DNA and gives the DNA polymerase a place to attach and begin synthesis of replicated DNA, extending the new strand from the 3’ end of the short RNA sequence

• This process is known as priming, and the short sequence is a primer

5’ 3’

3’ 5’

5’ PrimerPrimer 5’

G A G

C T C

C T G

C T G

DNA being replicated – replication of the new DNA strand proceeds from 3’ to 5’ along the template DNA, and since the two ‘backbones’ run in opposite directions, two primers are needed – one for each strand, each starting at the 3’ end of the origin of replication in its respective DNA strand

G A C C T C

Primers in the Lab• Primers can be made

synthetically and are used in DNA protocols such as SEQUENCING and the polymerase chain reaction (PCR)

• They are ordered from scientific suppliers and are diluted for use

• Primers are stored at -20oC

Primer stocksDiluted primers

Primers in the Lab, PCR

• In PCR reactions, specific primers are chosen to allow the amplification of desired fragments of DNA

• This can increase the quantity of target DNA by up to 108 times

• The rest of the DNA present in a sample is not amplified

Primers and PCR

• Here special primers have been designed to amplify a gene (CHD) found on bird sex chromosomes

• When the amplified DNA is run on a gel, only the target DNA is present in sufficient amounts to be visible

Note: Birds are different! Females are ZW and males ZZ. The gel above showstwo bands (Z and W) for females andone band for males– this is useful insexing birds such as parrots and kiwiwhere male and female look alike

Primers and Sequencing

• DNA sequencing also requires primers

• The DNA to be sequenced is usually first amplified with primers

• Specific primers are then used to allow incorporation of fluorescently labeled dideoxynucleotides into the newly synthesized DNA

• The labeled DNA is then analyzed with a sequencing machine and sequencing software

The insideof asequencer

Sequencing result

Primer Design

• Which came first- the chicken or the egg?

• If we know a DNA sequence, then primer design is fairly straightforward

• BUT how can we design a primer if the sequence isn’t known?

Making a primer from scratch• If a DNA sequence is unknown, then there are a number

of strategies for primer design• One involves inserting a piece of the DNA into bacterial

DNA of known sequence (cloning) using restriction enzymes, then using primers for the bacterial DNA to sequence the unknown DNA , then create primers for it.

• Sometimes the desired gene sequence is known for another organism. In this case primers would be based on the known DNA. Since DNA for equivalent genes can be very similar (highly conserved), this strategy is often successful

• Another is the determination of DNA sequence from a known amino acid sequence- this is fiddly because of DNA’s redundant code

• Other strategies exist – but you’re not graduate students!

Making a primer if you know a DNA sequence

• Primer design can be done with the aid of a computer programme- in this case, Primer 3, if a sequence is known

• Often, however, a researcher may choose to work manually from the print-out of a sequence

Primer Guidelines

• Primers are designed in pairs, one for each DNA strand, and are always written 5’-3’. They are complementary to strands of the relevant piece of DNA

• Primers should be at least 18 bases long (to be as specific as possible)

• They should contain about 40-60% G/C (why?)• Single base ‘runs’ of more than 4 bases in a row should

be avoided• There are many other considerations that guide

researchers in the development of good primers that give reliable PCR and sequencing results

Acknowledgements

Thanks to Dr. Leon Huynen, Massey University and Steve Chambers, University of Auckland

Compiled by Linda MacdonaldWhile on a RSNZ Science, Mathematics and Technology

Teacher Fellowship