Shutter Product Formation

Shutter Product Formation

• One allele/One STR site → Two peaks come with an additional one.

• The shutter product peak is 617-10% of the actual peak.

Shutter Product formation has to be avoided or ignored.

The reason behind shutter product formation is replication slippage. Replication slippage is of two types, can be formed or backward. Mostly the size is small of the shutter product in both cases. If there is both backward & forward slippage then:

There are two cases:

1. 

____ATTGCATGGC____

_____________________

• No repeats. 

• No shutter product formation.

• No replication Slippage.

Even after dissociation & re-association result will be the same.
2. 

___CGGCGGCGG___

___________________

• Repeats 

As both strands dissociate are chances of self-annealing.

GC

GG

CG

 GC

GG

_____CG_____

_____________

• Backward replication slippage.

• If template folds then it will be forward replication slippage.

From this, it is seen that shutter product is always scrolling in size and has to be ignored.

If there are different alleles for a site then there will be more chances of shutter product formation. (more copy number). Some STR sites may have 80 alleles as repeats will be more there will be more chances of shutter product formation.

Shutter product peak contributes less than 13%. If above 15% then it will not be ignored, it might not be shutter product formation.

How to avoid shutter Product Formation?

1. If such STR sites are taken which have large nucleotide repeats then the more will be the chance of shutter product formation. Mostly tetranucleotide repeats are in use. If a problem occurs even by using them then increase repeat size e.g pentanucleotide repeats or hexanucleotide repeats.

2. If possible then select imperfect repeats e.g four nucleotides are not repeating always mix repeats.

   1             2            3

                      CTTT    CTTT    CTTT  3 Repeats

   1             2           
                        CTTT    CTTT    CTTT  2.3 Repeats

As no chance of self-annealing so this site is more beneficial to be used as STR.

3. Take such DNA polymerase which has more processivity. It neither displaces the new strand not gets displaced. As there is such an enzyme that does not dissociate so there will be no shutter product formation.

Non-Template Addition:
Taq polymerase adds 🅰️ at the end of the product so product size changes. As some 🅰️ are added and some not so 2 peaks will come due to 2 products (due to addition of a single nucleotide) or it can be one peak with 2 heads.

Sometimes 🅰️ it is added & sometimes not in the product but we want the only a peak, either by the addition of A every time or by no addition every time. For this purpose, a sequence can be added at 5'end of primers i.e GTTCTT. Taq polymerase has a preference that if this sequence is present then it will necessarily add A so there will be 1005 chance of addition of 🅰️.

Extension time also has to be increased. When there will be more extension time then the probability will be more to form a product. Taq polymerase will cause addition at 3’end.
Another option is not to use polymerase. Such polymerase can be used which does not cause addiction-like T4 DNA polymerase or pfu polymerase. These will remove 🅰️ over changes. In the end, there will be such a product that does not have 🅰️ added.

With the addition of the GTTCTT sequence at 5'end of the primer, such restriction site should also be added which gives blunt end. The product is treated with the respective restriction endonuclease as a result of which a blunt end is created and a single peak is obtained. Even if there are some sticky ends then by heating with S1 nuclease single peak will be obtained.

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