Degenrative primer designing mannual method

1. Lab Assignment of Bioinformatics under the supervision of MAM
Misbah BILAL .Student name Zohaib HUSSAIN
Blast sequences
> Drosophila melanogaster
AACTGTCTGGTAATACTAGAGCATATACGTCAAAAACGCGCTAATTTAAAAGTCGGTGGCTTGCAAAGAA
ATAGCTTAATAAATTATGGAGGATTTTGAGAAAATTGAGAAGATTGGCGAGGGCACATATGGCGTGGTGT
ATAAGGGTCGCAACCGCCTGACGGGCCAAATTGTGGCAATGAAGAAAATCCGCTTGGAGTCCGACGACGA
AGGCGTTCCATCAACCGCGATCAGAGAAATTTCGTTGCTTAAGGAGTTGAAACATGAAAACATTGTCTGT
TTGGAGGATGTTTTGATGGAGGAGAACCGCATATACTTGATCTTTGAATTCCTATCGATGGACCTCAAGA
AATACATGGATTCGCTGCCAGTTGATAAGCACATGGAGAGTGAATTGGTCCGTAGCTATTTGTACCAAAT
AACTAGCGCCATTCTTTTCTGCCATCGTCGGCGAGTACTTCACCGTGATCTTAAGCCGCAGAACTTACTA
ATCGACAAGAGTGGCCTCATAAAAGTCGCCGACTTTGGACTTGGCCGATCCTTTGGCATTCCGGTGCGCA
TTTATACGCACGAGATTGTTACCTTGTGGTACAGAGCGCCGGAGGTGCTACTGGGTTCACCCCGGTATTC
CTGTCCCGTCGATATCTGGTCCATTGGATGCATATTCGCGGAGATGGCAACGAGAAAGCCGCTATTCCAG
GGTGACTCGGAAATTGACCAGTTGTTTAGAATGTTTAGAATTCTGAAAACACCTACCGAAGACATTTGGC
CGGGCGTTACTTCGCTACCCGACTATAAGAACACGTTCCCCTGCTGGTCCACGAACCAATTGACCAATCA
GTTAAAGAATCTCGATGCGAATGGTATTGATCTCATACAAAAGATGTTAATCTACGATCCAGTTCATCGC
ATTTCCGCCAAGGACATTTTGGAGCATCCCTATTTCAATGGTTTTCAATCGGGCTTAGTTCGAAATTAAC
GTTCGGTATTCTCGTTTGACTTTAACTAAGAATTTTAAAACAAGAGATCTTGGTATCTAATCTAAAGCAA
AATAGCCGTAAATAAAACTAAGGGTGTAAAAC
> Drosophila erecta GG23938
ATGGAGGATTTCGAGAAAATTGAGAAGATTGGCGAAGGCACATATGGTGTGGTATATAAGGGTCGCAACC
GACTGACGGGCCAAATTGTTGCGATGAAGAAAATTCGCTTGGAGTCCGACGACGAAGGCGTTCCATCAAC
AGCGATCAGAGAAATTTCGTTGCTCAAGGAGCTGAAACATGAAAACATTGTTTGTTTGGAGGATGTTCTG
ATGGAGGAGAATCGTATATACTTGATCTTTGAATTTCTATCGATGGACCTCAAGAAATACATGGATTCGC
TGCCAGTTGATAAGCACATGGAGAGTGAATTGGTTCGAAGCTATTTGTACCAAATAACCAGCGCCATTCT
ATTCTGCCATCGTCGGCGAGTACTGCACCGAGATCTTAAGCCGCAGAACTTACTTATCGACAAGAGTGGC
CTTATAAAAGTCGCCGACTTTGGACTTGGCCGATCCTTTGGCATTCCGGTGCGCATTTATACCCACGAGA
TCGTTACCTTGTGGTACAGAGCGCCGGAAGTGCTACTGGGATCACCCAGGTATTCCTGTCCCGTCGATAT
CTGGTCCATTGGGTGCATATTCGCGGAGATGGCAACGAGAAAGCCGCTGTTCCAGGGCGACTCGGAAATC
GACCAGTTATTCAGAATGTTCAGAATTCTTAAAACACCTACCGAAGACATTTGGCCGGGCGTTACTTCGC
TGCCCGACTATAAGAACACATTCCCCTGCTGGTCCACGAACCAATTGACCAACCAGTTGAAGAATCTCGA
TGAGAATGGTATTGATCTCATTCAAAAGATGTTAATCTACGATCCGGTTCATCGCATTTCCGCCAAGGAA
ATTTTGGAACACCCCTATTTCAATGGTTTTCAATCGGGCTTAGTTCGAAATTAA
> Drosophila yakuba
ATGGAGGATTTCGAGAAAATTGAGAAGATTGGCGAAGGCACATATGGAGTGGTATATAAGGGTCGCAACC
GCCTGACAGGCCAAATTGTGGCGATGAAGAAAATCCGCTTGGAGTCTGACGACGAAGGCGTTCCATCAAC
CGCGATCAGAGAAATTTCGTTGCTAAAGGAGCTGAAACATGAAAACATTGTCTGTTTGGAGGATGTTTTG
ATGGAGGAGAACCGCATATACTTGATCTTTGAATTCTTATCGATGGACCTTAAGAAATACATGGATTCTC
TGCCAGTTGATAAGCACATGGAGAGTGAATTGGTTCGAAGCTATTTGTACCAAATAACCAGCGCCATTCT
GTTCTGCCATCGTCGGCGAGTTCTTCACCGCGATCTTAAGCCGCAGAACTTACTTATCGACAAGAGTGGC
CTTATAAAAGTCGCCGACTTTGGACTGGGGCGATCCTTTGGCATTCCGGTGCGTATTTATACCCATGAGA
TCGTGACCTTGTGGTACAGAGCGCCGGAAGTGCTACTGGGATCACCCCGGTATTCCTGTCCCGTCGATAT
CTGGTCCATTGGGTGCATATTTGCGGAGATGGCAACGAGAAAACCGCTGTTCCAGGGCGACTCGGAGATC
GACCAGTTATTTAGAATGTTCAGGATTCTTAAAACACCTACCGAAGACATTTGGCCGGGCGTTACTTCGC
TGCCCGACTATAAGAATACGTTCCCCTGCTGGTCTACGAACCAATTAACCAACCAGTTGAAGAATCTCGA
TGCGAATGGTATTGATCTCATTCAAAAGATGTTAATTTACGATCCGGTTCATCGCATTTCCGCTAAGGAC
ATTTTGGAGCACCCCTATTTCAATGGTTTTCAATCGGGCTTAGTTCGAAATTA

2. > Drosophila simulans
ATGGAGGATTTCGAGAAAATTGAGAAGATTGGCGAGGGCACATATGGCGTGGTGTATAAGGGTCGCAATC
GCCTGACCGGCCAAATTGTGGCAATGAAGAAAATCCGCTTGGAGTCCGACGACGAAGGCGTTCCATCAAC
CGCGATCAGGGAAATTTCGTTGCTTAAGGAGTTGAAACATGAAAACATTGTCTGTTTGGAGGATGTTTTG
ATGGAGGAGAATCGCATATACTTGATCTTTGAATTCCTATCGATGGACCTCAAGAAATACATGGATTCGC
TGCCAGTTGATAAGCACATGGAGAGTGAATTGGTCCGTAGCTATTTGTACCAAATAACTAGCGCCATTCT
GTTCTGCCATCGTCGGCGAGTACTTCACCGTGATCTTAAGCCGCAGAACTTACTAATCGACAAGAGTGGC
CTCATAAAAGTCGCCGACTTTGGACTTGGCCGATCCTTTGGCATTCCGGTGCGCATTTATACGCACGAGA
TTGTGACCTTGTGGTACAGAGCGCCGGAGGTGCTACTGGGATCACCCCGAATTCTGAAAACACCTACCGA
AGACATTTGGCCGGGTGTTACTTCGCTACCCGACTATAAGAACACGTTCCCCTGCTGGTCCACAAACCAA
TTGACCAACCAGTTGAAGAATCTCGATGCGAATGGTATTGATCTCATTCAAAAGATGTTAATCTACGATC
CGGTTCATCGCATTTCCGCCAAGGACATTTTGGAGCATCCCTATTTCAATGGTTTTCAATCGGGCTTAGT
TCGAAATTAA
Blast result :
CLUSTAL 2.1 multiple sequence alignment
NOTE THE FORWARD AND REVERSE PRIMER ARE
HIGHLIGHTED BY RED COLOUR
Drosophila AACTGTCTGGTAATACTAGAGCATATACGTCAAAAACGCGCTAATTTAAAAGTCGGTGGC 60
simulans ------------------------------------------------------------
erecta ------------------------------------------------------------
yakuba ------------------------------------------------------------
Drosophila TTGCAAAGAAATAGCTTAATAAATTATGGAGGATTTTGAGAAAATTGAGAAGATTGGCGA 120
simulans -------------------------ATGGAGGATTTCGAGAAAATTGAGAAGATTGGCGA 35
erecta -------------------------ATGGAGGATTTCGAGAAAATTGAGAAGATTGGCGA 35
yakuba -------------------------ATGGAGGATTTCGAGAAAATTGAGAAGATTGGCGA 35
*********** ***********************

3. Drosophila GGGCACATATGGCGTGGTGTATAAGGGTCGCAACCGCCTGACGGGCCAAATTGTGGCAAT 180
simulans GGGCACATATGGCGTGGTGTATAAGGGTCGCAATCGCCTGACCGGCCAAATTGTGGCAAT 95
erecta AGGCACATATGGTGTGGTATATAAGGGTCGCAACCGACTGACGGGCCAAATTGTTGCGAT 95
yakuba AGGCACATATGGAGTGGTATATAAGGGTCGCAACCGCCTGACAGGCCAAATTGTGGCGAT 95
*********** ***** ************** ** ***** *********** ** **
Drosophila GAAGAAAATCCGCTTGGAGTCCGACGACGAAGGCGTTCCATCAACCGCGATCAGAGAAAT 240
simulans GAAGAAAATCCGCTTGGAGTCCGACGACGAAGGCGTTCCATCAACCGCGATCAGGGAAAT 155
erecta GAAGAAAATTCGCTTGGAGTCCGACGACGAAGGCGTTCCATCAACAGCGATCAGAGAAAT 155
yakuba GAAGAAAATCCGCTTGGAGTCTGACGACGAAGGCGTTCCATCAACCGCGATCAGAGAAAT 155
********* *********** *********************** ******** *****
Drosophila TTCGTTGCTTAAGGAGTTGAAACATGAAAACATTGTCTGTTTGGAGGATGTTTTGATGGA 300
simulans TTCGTTGCTTAAGGAGTTGAAACATGAAAACATTGTCTGTTTGGAGGATGTTTTGATGGA 215
erecta TTCGTTGCTCAAGGAGCTGAAACATGAAAACATTGTTTGTTTGGAGGATGTTCTGATGGA 215
yakuba TTCGTTGCTAAAGGAGCTGAAACATGAAAACATTGTCTGTTTGGAGGATGTTTTGATGGA 215
********* ****** ******************* *************** *******
Drosophila GGAGAACCGCATATACTTGATCTTTGAATTCCTATCGATGGACCTCAAGAAATACATGGA 360
simulans GGAGAATCGCATATACTTGATCTTTGAATTCCTATCGATGGACCTCAAGAAATACATGGA 275
erecta GGAGAATCGTATATACTTGATCTTTGAATTTCTATCGATGGACCTCAAGAAATACATGGA 275
yakuba GGAGAACCGCATATACTTGATCTTTGAATTCTTATCGATGGACCTTAAGAAATACATGGA 275
****** ** ******************** ************* **************
Drosophila TTCGCTGCCAGTTGATAAGCACATGGAGAGTGAATTGGTCCGTAGCTATTTGTACCAAAT 420
simulans TTCGCTGCCAGTTGATAAGCACATGGAGAGTGAATTGGTCCGTAGCTATTTGTACCAAAT 335
erecta TTCGCTGCCAGTTGATAAGCACATGGAGAGTGAATTGGTTCGAAGCTATTTGTACCAAAT 335
yakuba TTCTCTGCCAGTTGATAAGCACATGGAGAGTGAATTGGTTCGAAGCTATTTGTACCAAAT 335
*** *********************************** ** *****************
Drosophila AACTAGCGCCATTCTTTTCTGCCATCGTCGGCGAGTACTTCACCGTGATCTTAAGCCGCA 480
simulans AACTAGCGCCATTCTGTTCTGCCATCGTCGGCGAGTACTTCACCGTGATCTTAAGCCGCA 395
erecta AACCAGCGCCATTCTATTCTGCCATCGTCGGCGAGTACTGCACCGAGATCTTAAGCCGCA 395
yakuba AACCAGCGCCATTCTGTTCTGCCATCGTCGGCGAGTTCTTCACCGCGATCTTAAGCCGCA 395
*** *********** ******************** ** ***** **************
Drosophila GAACTTACTAATCGACAAGAGTGGCCTCATAAAAGTCGCCGACTTTGGACTTGGCCGATC 540
simulans GAACTTACTAATCGACAAGAGTGGCCTCATAAAAGTCGCCGACTTTGGACTTGGCCGATC 455
erecta GAACTTACTTATCGACAAGAGTGGCCTTATAAAAGTCGCCGACTTTGGACTTGGCCGATC 455
yakuba GAACTTACTTATCGACAAGAGTGGCCTTATAAAAGTCGCCGACTTTGGACTGGGGCGATC 455
********* ***************** *********************** ** *****
Drosophila CTTTGGCATTCCGGTGCGCATTTATACGCACGAGATTGTTACCTTGTGGTACAGAGCGCC 600
simulans CTTTGGCATTCCGGTGCGCATTTATACGCACGAGATTGTGACCTTGTGGTACAGAGCGCC 515
erecta CTTTGGCATTCCGGTGCGCATTTATACCCACGAGATCGTTACCTTGTGGTACAGAGCGCC 515
yakuba CTTTGGCATTCCGGTGCGTATTTATACCCATGAGATCGTGACCTTGTGGTACAGAGCGCC 515
****************** ******** ** ***** ** ********************
Drosophila GGAGGTGCTACTGGGTTCACCCCGGTATTCCTGTCCCGTCGATATCTGGTCCATTGGATG 660
simulans GGAGGTGCTACTGGGATCACCCCG------------------------------------ 539
erecta GGAAGTGCTACTGGGATCACCCAGGTATTCCTGTCCCGTCGATATCTGGTCCATTGGGTG 575
yakuba GGAAGTGCTACTGGGATCACCCCGGTATTCCTGTCCCGTCGATATCTGGTCCATTGGGTG 575
*** *********** ****** *
Drosophila CATATTCGCGGAGATGGCAACGAGAAAGCCGCTATTCCAGGGTGACTCGGAAATTGACCA 720
simulans ------------------------------------------------------------
erecta CATATTCGCGGAGATGGCAACGAGAAAGCCGCTGTTCCAGGGCGACTCGGAAATCGACCA 635
yakuba CATATTTGCGGAGATGGCAACGAGAAAACCGCTGTTCCAGGGCGACTCGGAGATCGACCA 635
Drosophila GTTGTTTAGAATGTTTAGAATTCTGAAAACACCTACCGAAGACATTTGGCCGGGCGTTAC 780
simulans ------------------AATTCTGAAAACACCTACCGAAGACATTTGGCCGGGTGTTAC 581
erecta GTTATTCAGAATGTTCAGAATTCTTAAAACACCTACCGAAGACATTTGGCCGGGCGTTAC 695

4. yakuba GTTATTTAGAATGTTCAGGATTCTTAAAACACCTACCGAAGACATTTGGCCGGGCGTTAC 695
***** ***************************** *****
Drosophila TTCGCTACCCGACTATAAGAACACGTTCCCCTGCTGGTCCACGAACCAATTGACCAATCA 840
simulans TTCGCTACCCGACTATAAGAACACGTTCCCCTGCTGGTCCACAAACCAATTGACCAACCA 641
erecta TTCGCTGCCCGACTATAAGAACACATTCCCCTGCTGGTCCACGAACCAATTGACCAACCA 755
yakuba TTCGCTGCCCGACTATAAGAATACGTTCCCCTGCTGGTCTACGAACCAATTAACCAACCA 755
****** ************** ** ************** ** ******** ***** **
Drosophila GTTAAAGAATCTCGATGCGAATGGTATTGATCTCATACAAAAGATGTTAATCTACGATCC 900
simulans GTTGAAGAATCTCGATGCGAATGGTATTGATCTCATTCAAAAGATGTTAATCTACGATCC 701
erecta GTTGAAGAATCTCGATGAGAATGGTATTGATCTCATTCAAAAGATGTTAATCTACGATCC 815
yakuba GTTGAAGAATCTCGATGCGAATGGTATTGATCTCATTCAAAAGATGTTAATTTACGATCC 815
*** ************* ****************** ************** ********
Drosophila AGTTCATCGCATTTCCGACTAGGACATTTTGGAGCATCCCTATTTCAATGGTTTTCAATC 960
simulans GGTTCATCGCATTTCCGACTAGGACATTTTGGAGCATCCCTATTTCAATGGTTTTCAATC 761
erecta GGTTCATCGCATTTCCGACTAGGAAATTTTGGAACACCCCTATTTCAATGGTTTTCAATC 875
yakuba GGTTCATCGCATTTCCGATTAGGACATTTTGGAGCACCCCTATTTCAATGGTTTTCAATC 875
***************** ***** ******** ** ***********************
Drosophila GGGCTTAGTTCGAAATTAACGTTCGGTATTCTCGTTTGACTTTAACTAAGAATTTTAAAA 1020
simulans GGGCTTAGTTCGAAATTAA----------------------------------------- 780
erecta GGGCTTAGTTCGAAATTAA----------------------------------------- 894
yakuba GGGCTTAGTTCGAAATTA------------------------------------------ 893
******************
Drosophila CAAGAGATCTTGGTATCTAATCTAAAGCAAAATAGCCGTAAATAAAACTAAGGGTGTAAA 1080
simulans ------------------------------------------------------------
erecta ------------------------------------------------------------
yakuba ------------------------------------------------------------
Drosophila AC 1082
simulans --
erecta --
yakuba --

5. ClustalW2 result
Phylogenetic tree

6. Forward primer sequences
Drosophila melanogaster =ATGGAGGAGAACCGCATAT
Drosophila simulans =ATGGAGGAGAATCGCATAT
Drosophila erecta =ATGGAGGAGAATCGTATAT
Drosophila yakuba =ATGGAGGAGAACCGCATAT
Forward primer
5’ATGGAGGAGAAYCGCATAT 3’ start codon is ATG
GC contents for Forward Primer
9/19 = 48%
NOTE (Primers with a 40-60% GC content ensure stable binding of primer)
Tm for Forward Primer :
Tm = 4(G+C) + 2(A+T)
=4(6+2)+2(7+5)
=32+24
Tm=56
REVERSE PRIMER
SEQ1=ACTAGGACATTTTGGAGCATCC
COMP=TGATCCTGTAAAACCTCGTAGG
SEQ2=ACTAGGACATTTTGGAGCATCC
COMP=TGATCCTGTAAAACCTCGTAGG
SEQ3=ACTAGGAAATTTTGGAACACCC
COMP=TGATCCTTTAAAACCTTGTGGG

7. SEQ4=ATTAGGACATTTTGGAGCACCC
COMP=TAACCTGTAAAACCTCGTGGG
COMPLEMNTRY SEQUENCES:
NOTE: these sequences are from
5’ 3’ direction
TGATCCTGTAAAACCTCGTAGG
TGATCCTGTAAAACCTCGTAGG
TGATCCTTTAAAACCTTGTGGG
TAATCCTGTAAAACCTCGTGGG
Reverse primer
5’TGATCCTGTAAAACCTCGTRGG ’3
3’GGRTGCTCCAAAATGTCCTAGT ‘5
TGA WILL ACT AS A STOP CODON
GC CONTENT
10/22 = 45%
NOTE (Primers with a 40-60% GC content ensure stable binding of primer)
Tm VALUE
Tm = 4(G+C) + 2(A+T)
=4(5+5)+2(5+6)
=40+22
=62