Summary: Giant oligos how-to

[Vivian Miao]

Here's a summary of responses (some posted to the group, 
some received by email) to my query about how to make a 
giant oligo in order to mutate a 226 bp fragment of dsDNA so 
that instead of being about 50% GC, it becomes 75% GC.  My 
conclusion from perusing the replies is that oligos 120 
bases or more can be made (but there are some things to 
watch out for), and that there are at least a few different 
ways to make my proposed experiment work (so I don't get to 
cash in the chips the 1st time things goes belly up ...). 
 Many thanks to all who contributed their time and 
experience!

=================Summary of original post=============
I'm thinking of ligating together several large "mutated" 
oligos that cover the 226 bp region.  Oligo #1 would 
correspond to position 1-50 of the top strand of the 
fragment, #2 would correspond to position 40-90 on the 
bottom strand; #3 would correspond to position 80-130 of the 
top strand, etc.  The oligos would be incubated together so 
the 3' overlaps can anneal, and then they would be primer 
extended and the nick left where the extension product and 
the next primer meet would be ligated.  Then maybe PCR to 
amplify up the correct product if it is rare before cloning.

Responses: general comments, specific methods and refs

====From: Alden Hackmann <darkstar at u.washington.edu>====
Sounds like it ought to work, on paper anyway.  It might be 
a bit tricky getting your overlapping oligo ends so that 
they don't anneal in the wrong places, which is a good case 
for doing the stepwise addition route rather than the 
quicker and dirtier one-pot-of-soup route.
==============From: WILLIS at BCRSSU.AGR.CA===========
I can send you a short (one page) protocol I developed for 
constructing synthetic genes. I once worked for a 
pharmaceutical company and had to routinely put synthetic 
genes together.  (Les' protocol describes using oligos that 
anneal to form ds DNA fragments with single stranded tails 
which are then are assembled to make the full size 
construct.  It includes operational details on how to make 
the assembly work.  It looks very helpful, and Les sent it 
the same day I asked my question!).
==============From: Jon R Sayers Univ of Wales========
....remember chemical synthesis is not as good as Nature.  a 
small %age will carry eg deaminations, T-C modifications 
etc.  If you want a specific sequence at the end of the day 
you might just try the standard shotgun Klenow approach with 
eg 50-70 mers, thus:
NNNNNNNNNN(5'P)-NN(50 orso)NNNNNNNNNN (5'P)NNNNNNNNNNNNN
        3'OH-NNNNNNNNNNNN(5'-P)      3'OHNNNNNNN(5'P)   NNNN
The lower oligos are ca 15 nucleotides, the upper 50-70. The 
bottom strand is produced using 5'-phosphorylated oligos and 
eg T7 polymerase. You can incorporate res. sites etc at the 
ends for cloning. The PCR techniques may be dandy but whatch 
out for artifacts introduced by multiple cycles. The above 
method needs only one cycle.
=====From: Michael Kurilla <mgk2r at virginia.edu>========
ABI PCRMATE oligo synthesizer ... showed the output of a 
186mer with about 5-10% yield.  Since you can purify the 
product over a trityl column, most of the early failures 
would be removed. since you'd need to make both strands 
(unless it was perfectly palindromic), I would make two 
smaller oligos (120 - 130 bases) with about 20 bases of 
overlap, anneal, and fillin with either Klenow or T4 DNA 
polymerase.
==From: Graham Atherton<GRGGTA at UK.AC.MAN.CR.PICR>==
I have successfully made 120bp oligos, annealed them and 
cloned them into pUC-based vectors. The synthesis yielded 
approx 0.5mg DNA using standard (0.2 micromole) columns 
followed by cleaning through NENSORB affinity columns - 
again, in the same way I do all of my oligos. I believe that 
200bp oligos are possible, the only provisio being that the 
yield of the full-length product gets lower as you add more 
bases because the efficiency of the reaction is only around 
99.5% - but improvements on this may have  already been 
made. The upper size limit of the oligo is defined by the  
'pore size' in the synthesis column. It may be with 
contacting an oligo synthesis company to see if you could 
get your 250bp oligos made in one go!
========From: <sjszarka at acs.ucalgary.ca>===========
I am responsible for the oligo machine in our lab and about 
a year ago one of the other grad students needed to create a 
90 amino acid peptide with the codon usage of a different 
organism. We did this by synthesizing two primers; one about 
170 nts and one about 150 nts. The two primers had about 25 
nts of overlap. Since the sythesis is never 100% for each 
base added, we anticipated that the amount of full length 
primer for the 150 and 170 mers would probably be quit low. 
To select only full length products we also synthesized two 
18 -20 mers to the ends of the large primers.
	PCR was run for I believe 10-20 cycles with the two 
large primers to create the templates for the smaller end 
primers. Which were then added and used to select only the 
full length templates. Thefinal product was then cloned and 
sequenced.
	The idea of giant oligos is feasible but because of 
the low yieldof full length oligos of that size some way of 
selecting or purifying out full length products is 
necessary.
	Columns with a larger pore size were specially ordered 
for the large synthesis.The machine ran around the clock, 
which meant that someone had to come in to the lab in the 
middle of the night to replenish chemicals and check the 
progress.  Nothing I would look forward to doing any time 
soon.
====From: Bob DeLisio <delisior at rnisd0.DNET.roche.com>=====
A RAPID METHOD FOR THE CONSTRUCTION OF SYNTHETIC GENES
USING THE POLYMERASE CHAIN REACTION.  Benchmark, Biotech
niques, Vol.9,(3), p.298-300.  (This paper describes synthesis of 
a large construct via a scheme like the one outlined in the 
original question.  One cheering note was that they did not 
need to purify their oligos <hallelujah> for their 
construction to work). 
======From: Keith Robison <robison at biosun.harvard.edu>====
... a number of references ... two "classic" examples are 
Khorana's synthesis of tRNA in the early 70's and the Boyer 
group's synthesis of a growth hormone gene in the late 70's.
=======================From: G W Chacko================
on the subject of classic examples: Total Synthesis Of A 
Gene For Bovine Rhodopsin.  Ferretti et al. PNAS. 83:599-
603. (1986)  (ligated 72 oligos to make a 1072 bp fragment).
=====From: "Dr. A. Rosenthal" <arosenth at med.cam.ac.uk>=====
(Dr. Rosenthal wrote that he knew of folks who were planning 
to use this method for gene therapy, and who had already 
obtained the oligos.  Dr. Rosenthal offered to check for 
follow up to how the project worked out). 
======================From:John Hachey================
DiDonato et al.1993. A Method for synthesizing genes and
 cdnas by the polymerase chain reaction. Anal. Biochem.
 212:291-293.