1. Describe how you would adjust the amplicon sizes for 2 STR loci with similar

ID: 167111 • Letter: 1

Question

1. Describe how you would adjust the amplicon sizes for 2 STR loci with similar repeat ranges so that you can resolve them in a single gel lane (e.g. TPOX alleles 6-16; THO1 alleles 4-15).

2. You have the following 4 STR loci with the described allelic size range. You need to separate them using ROX and JOE-tagged primers, 2 fluorophores. Decide which loci will be tagged with which dye (2 loci for each dye), and order them in size from high to low (e.g. ROX: Penta E, D5S818).

Loci:

FGA - allelic range: 308-464 nt (alleles 12-51)

CSF1PO - allelic range: 317-361 nt (alleles 5-16)

TPOX - allelic range: 254-302 nt (alleles 4-16)

vWA - allelic range: 152-212 (alleles 10-25)

Explanation / Answer

Q.No 1

Amplicon size must be in the range between 200-1000bp. The particular polymerization of the enzyme found in the response ought to be satisfactory to copy the amplicon in its entirety, hence for long amplicons, you should enhance the extension cycle time. The amplicon sizes in base pairs (bp) are different for a lot of overlapping loci of these multiplex PCR systems. The particular resulting amplicon size connected with a pair of STR loci these kinds of as TPOX allele 9 and TH01 allele 11. Null alleles undoubtedly are a problem simply because this could result in a false-negative or maybe wrong different connected with two samples that come from a typical source. A base pair change throughout the DNA template at the PCR-primer-binding place could interrupt primer hybridization and cause a disappointment for you to boost and recognize a current allele.

Q.No 2

Locus

Size range (bP)

Location of chromosome

Labelled Dye

CSF1PO

281–317

(AGAT)n

JOE

FGA

219–267

(TTTC)3 TTTT TTCT (CTTT)n CTCC (TTCC)2

ROX

TPOX

218–242

(AATG)n

JOE

vWA

157–197

TCTA (TCTG)3-4 (TCTA)n

ROX