v:* {behavior:url(#default#VML);}
o:* {behavior:url(#default#VML);}
w:* {behavior:url(#default#VML);}
.shape {behavior:url(#default#VML);}
Normal
0
false
false
false
false
EN-GB
X-NONE
AR-SA
/* Style Definitions */
table.MsoNormalTable
{mso-style-name:”Table Normal”;
mso-tstyle-rowband-size:0;
mso-tstyle-colband-size:0;
mso-style-noshow:yes;
mso-style-priority:99;
mso-style-parent:””;
mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
mso-para-margin:0cm;
mso-para-margin-bottom:.0001pt;
mso-pagination:widow-orphan;
font-size:11.0pt;
font-family:”Calibri”,sans-serif;
mso-ansi-language:EN-GB;}
table.MsoTableGrid
{mso-style-name:”Table Grid”;
mso-tstyle-rowband-size:0;
mso-tstyle-colband-size:0;
mso-style-priority:59;
mso-style-unhide:no;
border:solid black 1.0pt;
mso-border-themecolor:text1;
mso-border-alt:solid black .5pt;
mso-border-themecolor:text1;
mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
mso-border-insideh:.5pt solid black;
mso-border-insideh-themecolor:text1;
mso-border-insidev:.5pt solid black;
mso-border-insidev-themecolor:text1;
mso-para-margin:0cm;
mso-para-margin-bottom:.0001pt;
mso-pagination:widow-orphan;
font-size:11.0pt;
font-family:”Calibri”,sans-serif;
mso-ascii-font-family:Calibri;
mso-ascii-theme-font:minor-latin;
mso-fareast-font-family:”Times New Roman”;
mso-fareast-theme-font:minor-fareast;
mso-hansi-font-family:Calibri;
mso-hansi-theme-font:minor-latin;
mso-ansi-language:EN-GB;
mso-fareast-language:EN-GB;}
table.MsoTableColorfulGridAccent1
{mso-style-name:”Colorful Grid – Accent 1″;
mso-tstyle-rowband-size:1;
mso-tstyle-colband-size:1;
mso-style-priority:73;
mso-style-unhide:no;
mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
mso-border-insideh:.5pt solid white;
mso-border-insideh-themecolor:background1;
mso-tstyle-shading:#DBE5F1;
mso-tstyle-shading-themecolor:accent1;
mso-tstyle-shading-themetint:51;
mso-para-margin:0cm;
mso-para-margin-bottom:.0001pt;
mso-pagination:widow-orphan;
font-size:11.0pt;
font-family:”Calibri”,sans-serif;
mso-ascii-font-family:Calibri;
mso-ascii-theme-font:minor-latin;
mso-hansi-font-family:Calibri;
mso-hansi-theme-font:minor-latin;
color:black;
mso-themecolor:text1;
mso-ansi-language:EN-GB;}
table.MsoTableColorfulGridAccent1FirstRow
{mso-style-name:”Colorful Grid – Accent 1″;
mso-table-condition:first-row;
mso-style-priority:73;
mso-style-unhide:no;
mso-tstyle-shading:#B8CCE4;
mso-tstyle-shading-themecolor:accent1;
mso-tstyle-shading-themetint:102;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableColorfulGridAccent1LastRow
{mso-style-name:”Colorful Grid – Accent 1″;
mso-table-condition:last-row;
mso-style-priority:73;
mso-style-unhide:no;
mso-tstyle-shading:#B8CCE4;
mso-tstyle-shading-themecolor:accent1;
mso-tstyle-shading-themetint:102;
color:black;
mso-themecolor:text1;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableColorfulGridAccent1FirstCol
{mso-style-name:”Colorful Grid – Accent 1″;
mso-table-condition:first-column;
mso-style-priority:73;
mso-style-unhide:no;
mso-tstyle-shading:#365F91;
mso-tstyle-shading-themecolor:accent1;
mso-tstyle-shading-themeshade:191;
color:white;
mso-themecolor:background1;}
table.MsoTableColorfulGridAccent1LastCol
{mso-style-name:”Colorful Grid – Accent 1″;
mso-table-condition:last-column;
mso-style-priority:73;
mso-style-unhide:no;
mso-tstyle-shading:#365F91;
mso-tstyle-shading-themecolor:accent1;
mso-tstyle-shading-themeshade:191;
color:white;
mso-themecolor:background1;}
table.MsoTableColorfulGridAccent1OddColumn
{mso-style-name:”Colorful Grid – Accent 1″;
mso-table-condition:odd-column;
mso-style-priority:73;
mso-style-unhide:no;
mso-tstyle-shading:#A7BFDE;
mso-tstyle-shading-themecolor:accent1;
mso-tstyle-shading-themetint:127;}
table.MsoTableColorfulGridAccent1OddRow
{mso-style-name:”Colorful Grid – Accent 1″;
mso-table-condition:odd-row;
mso-style-priority:73;
mso-style-unhide:no;
mso-tstyle-shading:#A7BFDE;
mso-tstyle-shading-themecolor:accent1;
mso-tstyle-shading-themetint:127;}
table.MsoTableMediumShading1Accent5
{mso-style-name:”Medium Shading 1 – Accent 5″;
mso-tstyle-rowband-size:1;
mso-tstyle-colband-size:1;
mso-style-priority:63;
mso-style-unhide:no;
border:solid #78C0D4 1.0pt;
mso-border-themecolor:accent5;
mso-border-themetint:191;
mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
mso-border-insideh:1.0pt solid #78C0D4;
mso-border-insideh-themecolor:accent5;
mso-border-insideh-themetint:191;
mso-para-margin:0cm;
mso-para-margin-bottom:.0001pt;
mso-pagination:widow-orphan;
font-size:11.0pt;
font-family:”Calibri”,sans-serif;
mso-ascii-font-family:Calibri;
mso-ascii-theme-font:minor-latin;
mso-hansi-font-family:Calibri;
mso-hansi-theme-font:minor-latin;
mso-ansi-language:EN-GB;}
table.MsoTableMediumShading1Accent5FirstRow
{mso-style-name:”Medium Shading 1 – Accent 5″;
mso-table-condition:first-row;
mso-style-priority:63;
mso-style-unhide:no;
mso-tstyle-shading:#4BACC6;
mso-tstyle-shading-themecolor:accent5;
mso-tstyle-border-top:1.0pt solid #78C0D4;
mso-tstyle-border-top-themecolor:accent5;
mso-tstyle-border-top-themetint:191;
mso-tstyle-border-left:1.0pt solid #78C0D4;
mso-tstyle-border-left-themecolor:accent5;
mso-tstyle-border-left-themetint:191;
mso-tstyle-border-bottom:1.0pt solid #78C0D4;
mso-tstyle-border-bottom-themecolor:accent5;
mso-tstyle-border-bottom-themetint:191;
mso-tstyle-border-right:1.0pt solid #78C0D4;
mso-tstyle-border-right-themecolor:accent5;
mso-tstyle-border-right-themetint:191;
mso-tstyle-border-insideh:cell-none;
mso-tstyle-border-insidev:cell-none;
mso-para-margin-top:0cm;
mso-para-margin-bottom:0cm;
mso-para-margin-bottom:.0001pt;
line-height:normal;
color:white;
mso-themecolor:background1;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableMediumShading1Accent5LastRow
{mso-style-name:”Medium Shading 1 – Accent 5″;
mso-table-condition:last-row;
mso-style-priority:63;
mso-style-unhide:no;
mso-tstyle-border-top:2.25pt double #78C0D4;
mso-tstyle-border-top-themecolor:accent5;
mso-tstyle-border-top-themetint:191;
mso-tstyle-border-left:1.0pt solid #78C0D4;
mso-tstyle-border-left-themecolor:accent5;
mso-tstyle-border-left-themetint:191;
mso-tstyle-border-bottom:1.0pt solid #78C0D4;
mso-tstyle-border-bottom-themecolor:accent5;
mso-tstyle-border-bottom-themetint:191;
mso-tstyle-border-right:1.0pt solid #78C0D4;
mso-tstyle-border-right-themecolor:accent5;
mso-tstyle-border-right-themetint:191;
mso-tstyle-border-insideh:cell-none;
mso-tstyle-border-insidev:cell-none;
mso-para-margin-top:0cm;
mso-para-margin-bottom:0cm;
mso-para-margin-bottom:.0001pt;
line-height:normal;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableMediumShading1Accent5FirstCol
{mso-style-name:”Medium Shading 1 – Accent 5″;
mso-table-condition:first-column;
mso-style-priority:63;
mso-style-unhide:no;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableMediumShading1Accent5LastCol
{mso-style-name:”Medium Shading 1 – Accent 5″;
mso-table-condition:last-column;
mso-style-priority:63;
mso-style-unhide:no;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableMediumShading1Accent5OddColumn
{mso-style-name:”Medium Shading 1 – Accent 5″;
mso-table-condition:odd-column;
mso-style-priority:63;
mso-style-unhide:no;
mso-tstyle-shading:#D2EAF1;
mso-tstyle-shading-themecolor:accent5;
mso-tstyle-shading-themetint:63;}
table.MsoTableMediumShading1Accent5OddRow
{mso-style-name:”Medium Shading 1 – Accent 5″;
mso-table-condition:odd-row;
mso-style-priority:63;
mso-style-unhide:no;
mso-tstyle-shading:#D2EAF1;
mso-tstyle-shading-themecolor:accent5;
mso-tstyle-shading-themetint:63;
mso-tstyle-border-insideh:cell-none;
mso-tstyle-border-insidev:cell-none;}
table.MsoTableMediumShading1Accent5EvenRow
{mso-style-name:”Medium Shading 1 – Accent 5″;
mso-table-condition:even-row;
mso-style-priority:63;
mso-style-unhide:no;
mso-tstyle-border-insideh:cell-none;
mso-tstyle-border-insidev:cell-none;}
table.MsoTableLightGridAccent6
{mso-style-name:”Light Grid – Accent 6″;
mso-tstyle-rowband-size:1;
mso-tstyle-colband-size:1;
mso-style-priority:62;
mso-style-unhide:no;
border:solid #F79646 1.0pt;
mso-border-themecolor:accent6;
mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
mso-border-insideh:1.0pt solid #F79646;
mso-border-insideh-themecolor:accent6;
mso-border-insidev:1.0pt solid #F79646;
mso-border-insidev-themecolor:accent6;
mso-para-margin:0cm;
mso-para-margin-bottom:.0001pt;
mso-pagination:widow-orphan;
font-size:11.0pt;
font-family:”Calibri”,sans-serif;
mso-ascii-font-family:Calibri;
mso-ascii-theme-font:minor-latin;
mso-hansi-font-family:Calibri;
mso-hansi-theme-font:minor-latin;
mso-ansi-language:EN-GB;}
table.MsoTableLightGridAccent6FirstRow
{mso-style-name:”Light Grid – Accent 6″;
mso-table-condition:first-row;
mso-style-priority:62;
mso-style-unhide:no;
mso-tstyle-border-top:1.0pt solid #F79646;
mso-tstyle-border-top-themecolor:accent6;
mso-tstyle-border-left:1.0pt solid #F79646;
mso-tstyle-border-left-themecolor:accent6;
mso-tstyle-border-bottom:2.25pt solid #F79646;
mso-tstyle-border-bottom-themecolor:accent6;
mso-tstyle-border-right:1.0pt solid #F79646;
mso-tstyle-border-right-themecolor:accent6;
mso-tstyle-border-insideh:cell-none;
mso-tstyle-border-insidev:1.0pt solid #F79646;
mso-tstyle-border-insidev-themecolor:accent6;
mso-para-margin-top:0cm;
mso-para-margin-bottom:0cm;
mso-para-margin-bottom:.0001pt;
line-height:normal;
font-family:”Cambria”,serif;
mso-ascii-font-family:Cambria;
mso-ascii-theme-font:major-latin;
mso-fareast-font-family:”Times New Roman”;
mso-fareast-theme-font:major-fareast;
mso-hansi-font-family:Cambria;
mso-hansi-theme-font:major-latin;
mso-bidi-font-family:”Times New Roman”;
mso-bidi-theme-font:major-bidi;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableLightGridAccent6LastRow
{mso-style-name:”Light Grid – Accent 6″;
mso-table-condition:last-row;
mso-style-priority:62;
mso-style-unhide:no;
mso-tstyle-border-top:2.25pt double #F79646;
mso-tstyle-border-top-themecolor:accent6;
mso-tstyle-border-left:1.0pt solid #F79646;
mso-tstyle-border-left-themecolor:accent6;
mso-tstyle-border-bottom:1.0pt solid #F79646;
mso-tstyle-border-bottom-themecolor:accent6;
mso-tstyle-border-right:1.0pt solid #F79646;
mso-tstyle-border-right-themecolor:accent6;
mso-tstyle-border-insideh:cell-none;
mso-tstyle-border-insidev:1.0pt solid #F79646;
mso-tstyle-border-insidev-themecolor:accent6;
mso-para-margin-top:0cm;
mso-para-margin-bottom:0cm;
mso-para-margin-bottom:.0001pt;
line-height:normal;
font-family:”Cambria”,serif;
mso-ascii-font-family:Cambria;
mso-ascii-theme-font:major-latin;
mso-fareast-font-family:”Times New Roman”;
mso-fareast-theme-font:major-fareast;
mso-hansi-font-family:Cambria;
mso-hansi-theme-font:major-latin;
mso-bidi-font-family:”Times New Roman”;
mso-bidi-theme-font:major-bidi;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableLightGridAccent6FirstCol
{mso-style-name:”Light Grid – Accent 6″;
mso-table-condition:first-column;
mso-style-priority:62;
mso-style-unhide:no;
font-family:”Cambria”,serif;
mso-ascii-font-family:Cambria;
mso-ascii-theme-font:major-latin;
mso-fareast-font-family:”Times New Roman”;
mso-fareast-theme-font:major-fareast;
mso-hansi-font-family:Cambria;
mso-hansi-theme-font:major-latin;
mso-bidi-font-family:”Times New Roman”;
mso-bidi-theme-font:major-bidi;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableLightGridAccent6LastCol
{mso-style-name:”Light Grid – Accent 6″;
mso-table-condition:last-column;
mso-style-priority:62;
mso-style-unhide:no;
mso-tstyle-border-top:1.0pt solid #F79646;
mso-tstyle-border-top-themecolor:accent6;
mso-tstyle-border-left:1.0pt solid #F79646;
mso-tstyle-border-left-themecolor:accent6;
mso-tstyle-border-bottom:1.0pt solid #F79646;
mso-tstyle-border-bottom-themecolor:accent6;
mso-tstyle-border-right:1.0pt solid #F79646;
mso-tstyle-border-right-themecolor:accent6;
font-family:”Cambria”,serif;
mso-ascii-font-family:Cambria;
mso-ascii-theme-font:major-latin;
mso-fareast-font-family:”Times New Roman”;
mso-fareast-theme-font:major-fareast;
mso-hansi-font-family:Cambria;
mso-hansi-theme-font:major-latin;
mso-bidi-font-family:”Times New Roman”;
mso-bidi-theme-font:major-bidi;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableLightGridAccent6OddColumn
{mso-style-name:”Light Grid – Accent 6″;
mso-table-condition:odd-column;
mso-style-priority:62;
mso-style-unhide:no;
mso-tstyle-shading:#FDE4D0;
mso-tstyle-shading-themecolor:accent6;
mso-tstyle-shading-themetint:63;
mso-tstyle-border-top:1.0pt solid #F79646;
mso-tstyle-border-top-themecolor:accent6;
mso-tstyle-border-left:1.0pt solid #F79646;
mso-tstyle-border-left-themecolor:accent6;
mso-tstyle-border-bottom:1.0pt solid #F79646;
mso-tstyle-border-bottom-themecolor:accent6;
mso-tstyle-border-right:1.0pt solid #F79646;
mso-tstyle-border-right-themecolor:accent6;}
table.MsoTableLightGridAccent6OddRow
{mso-style-name:”Light Grid – Accent 6″;
mso-table-condition:odd-row;
mso-style-priority:62;
mso-style-unhide:no;
mso-tstyle-shading:#FDE4D0;
mso-tstyle-shading-themecolor:accent6;
mso-tstyle-shading-themetint:63;
mso-tstyle-border-top:1.0pt solid #F79646;
mso-tstyle-border-top-themecolor:accent6;
mso-tstyle-border-left:1.0pt solid #F79646;
mso-tstyle-border-left-themecolor:accent6;
mso-tstyle-border-bottom:1.0pt solid #F79646;
mso-tstyle-border-bottom-themecolor:accent6;
mso-tstyle-border-right:1.0pt solid #F79646;
mso-tstyle-border-right-themecolor:accent6;
mso-tstyle-border-insidev:1.0pt solid #F79646;
mso-tstyle-border-insidev-themecolor:accent6;}
table.MsoTableLightGridAccent6EvenRow
{mso-style-name:”Light Grid – Accent 6″;
mso-table-condition:even-row;
mso-style-priority:62;
mso-style-unhide:no;
mso-tstyle-border-top:1.0pt solid #F79646;
mso-tstyle-border-top-themecolor:accent6;
mso-tstyle-border-left:1.0pt solid #F79646;
mso-tstyle-border-left-themecolor:accent6;
mso-tstyle-border-bottom:1.0pt solid #F79646;
mso-tstyle-border-bottom-themecolor:accent6;
mso-tstyle-border-right:1.0pt solid #F79646;
mso-tstyle-border-right-themecolor:accent6;
mso-tstyle-border-insidev:1.0pt solid #F79646;
mso-tstyle-border-insidev-themecolor:accent6;}
table.MsoTableMediumShading1Accent6
{mso-style-name:”Medium Shading 1 – Accent 6″;
mso-tstyle-rowband-size:1;
mso-tstyle-colband-size:1;
mso-style-priority:63;
mso-style-unhide:no;
border:solid #F9B074 1.0pt;
mso-border-themecolor:accent6;
mso-border-themetint:191;
mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
mso-border-insideh:1.0pt solid #F9B074;
mso-border-insideh-themecolor:accent6;
mso-border-insideh-themetint:191;
mso-para-margin:0cm;
mso-para-margin-bottom:.0001pt;
mso-pagination:widow-orphan;
font-size:11.0pt;
font-family:”Calibri”,sans-serif;
mso-ascii-font-family:Calibri;
mso-ascii-theme-font:minor-latin;
mso-hansi-font-family:Calibri;
mso-hansi-theme-font:minor-latin;
mso-ansi-language:EN-GB;}
table.MsoTableMediumShading1Accent6FirstRow
{mso-style-name:”Medium Shading 1 – Accent 6″;
mso-table-condition:first-row;
mso-style-priority:63;
mso-style-unhide:no;
mso-tstyle-shading:#F79646;
mso-tstyle-shading-themecolor:accent6;
mso-tstyle-border-top:1.0pt solid #F9B074;
mso-tstyle-border-top-themecolor:accent6;
mso-tstyle-border-top-themetint:191;
mso-tstyle-border-left:1.0pt solid #F9B074;
mso-tstyle-border-left-themecolor:accent6;
mso-tstyle-border-left-themetint:191;
mso-tstyle-border-bottom:1.0pt solid #F9B074;
mso-tstyle-border-bottom-themecolor:accent6;
mso-tstyle-border-bottom-themetint:191;
mso-tstyle-border-right:1.0pt solid #F9B074;
mso-tstyle-border-right-themecolor:accent6;
mso-tstyle-border-right-themetint:191;
mso-tstyle-border-insideh:cell-none;
mso-tstyle-border-insidev:cell-none;
mso-para-margin-top:0cm;
mso-para-margin-bottom:0cm;
mso-para-margin-bottom:.0001pt;
line-height:normal;
color:white;
mso-themecolor:background1;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableMediumShading1Accent6LastRow
{mso-style-name:”Medium Shading 1 – Accent 6″;
mso-table-condition:last-row;
mso-style-priority:63;
mso-style-unhide:no;
mso-tstyle-border-top:2.25pt double #F9B074;
mso-tstyle-border-top-themecolor:accent6;
mso-tstyle-border-top-themetint:191;
mso-tstyle-border-left:1.0pt solid #F9B074;
mso-tstyle-border-left-themecolor:accent6;
mso-tstyle-border-left-themetint:191;
mso-tstyle-border-bottom:1.0pt solid #F9B074;
mso-tstyle-border-bottom-themecolor:accent6;
mso-tstyle-border-bottom-themetint:191;
mso-tstyle-border-right:1.0pt solid #F9B074;
mso-tstyle-border-right-themecolor:accent6;
mso-tstyle-border-right-themetint:191;
mso-tstyle-border-insideh:cell-none;
mso-tstyle-border-insidev:cell-none;
mso-para-margin-top:0cm;
mso-para-margin-bottom:0cm;
mso-para-margin-bottom:.0001pt;
line-height:normal;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableMediumShading1Accent6FirstCol
{mso-style-name:”Medium Shading 1 – Accent 6″;
mso-table-condition:first-column;
mso-style-priority:63;
mso-style-unhide:no;
mso-ansi-font-weight:bold;
mso-bidi-font-weight:bold;}
table.MsoTableMediumShading1Accent6LastCol
{mso-style-name:”Medium Shading 1 – Accent 6″;
mso-table-condition:last-column;
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Assignment: Write up your PTC Genotype practical and the data you/all generated, using the format of a short scientific paper (approx. 3000words)
The assignment should include:
1. Title&Abstract (200words) (15 %)
Title/ PTC Genotype using the rustication enzyme (Fnu4H1).
2. Introduction: (500 words) (10 %)
Background of the study & hypothesis (taste perception/genetic link/PTC gene polymorphisms).
3. Methods
Extraction of DNA from cheek cells
The PCR is extremely sensitive to contamination. Thus, during the experiment should be kept sterile as well as wore gloves to prevent his from happening. During this experiment, a sterile wooden splint was performed to scrap loose buccal cells from the inside cheek. The cells were then placed away from the wooden loop using the sterile loop and swiveled into a 1.5 ml eppendorf tube containing 350 µl 5% Chelex suspension. protinase k 4µl was added (stock solution; 10mg/ml) which removed protein contamination. The sample was then incubated at 56ºC for 30 minutes and then vortexed for 10 seconds and then centrifuged at 13,000 rpm for 20 seconds. The tube was placed in a heating block at 98ºC for 15 minutes to anneal DNA from the proteins. The tube was again vortexed for 10 seconds and then centrifuged at maximum speed for 3 minutes. The supernatant (top layer) which had buccal cells DNA was transferred to a sterile 1.5ml eppendorf tube which was labelled and kept in ice to preserve the DNA. However, before it was stored in ice check the quantity of DNA in the sample it was by taking 5ul and then placed a sterile 0.5 ml eppendorf tube and was done the measurement of DNA quantity through on Nano-drop nucleic acid measurement machine.
PCR reaction
The micro eppendorf tube (PCR tube) was provided for Each student that had white bead known as Pharmacia PCR bead which contains the PCR reaction components. However, Master mix lacks the template DNA or amplification primers. Therefore, Master mix (43.5µl) containing both forward (5’AACTGGCAGATTAAAGATCTCAATTTA
T3’) and reverse (5’AACACAAACCATCACCCCTATTTT3’) primers was added to (6.5µl) of the template DNA in the PCR tube. The sample was then mixed to dissolve gather liquid content to the bottom and placed in the thermal cycler using the following program which done by module leader:
94˚C 4 minutes
|
|
55˚C 40 seconds
72˚C 40 seconds 40 cycles
94˚C 40 seconds
55˚C 5 minutes
72˚C 5 minutes
Restriction fragment length polymorphism analysis of PTC genotyping
After the PCR process was done, 20µl of PCR component mixture was added into a 0.5 ml eppendorf tube which had already10 µl of restriction master mix (Fnu4H1). Further, the Fnu4H1 had been prepared earlier and stored on ice. The mixture which totalled 30µl volume was centrifuged briefly and then placed into a heating block of 37°C minimum for 2 hours and the original PCR tube with 30µl remaining, placed back to the ice.
Gel electrophoresis
2% agarose gel, submerged in Tris-Borate-EDTA buffer was used to load both digested, undigested DNA and 8-10µl of 100bp molecular weight marker was loaded to the first well. Then two sets of samples (digested, undigested) were prepared. 8µl of loading buffer was added to both digested and undigested and then mix plus spin and loaded to the gel well.
The gel was prepared with ethidium bromide (0.5µg) Then, the gel was electrophoresed at 90 voltages for 45 minutes. Finally, the gel was visualised using a UV transilluminator and both the taster and non-tasters band indicated.
4. Results (no word count)
Figure 1: Illustrates PCR inferred results for PTC gene. 2% gel electrophoresis of digested and undigested DNA sample for PCR genotyping. Lane M is a 100 bp ladder. lane 3 is digested DNA sample using restriction master mix (Fnu4H1) which was provided during practical and lane 4 has undigested sample as indicated by asterisks to differentiate from DNA samples of other students where both lanes 6 and 7 are presenting author sample.
Table 1: Shows the distance of 100bp marker and logarithmic results Using Excel sheet. The distances of DNA migration on gel was measured using a ruler and estimated size of each fragment of DNA was then calculated shown in table 2.
|
Molecular weight |
Log of molecular weight |
Fragments Distance (mm) |
|
1000 |
3 |
9 |
|
900 |
2.95 |
9 |
|
800 |
2.90 |
10 |
|
700 |
2.85 |
11 |
|
600 |
2.78 |
12 |
|
500 |
2.70 |
13 |
|
400 |
2.60 |
14 |
|
300 |
2.48 |
16 |
|
200 |
2.30 |
18 |
|
100 |
2 |
20 |
Figure 2. Shows standard curve of molecular weight ladder against the fragment distance, the log of each band of 100bp marker was calculated and standard calibration curve was plotted as shown in figure 2.
Table 2: Explains the calculation of author fragment that observed in the gel which was in lane 3 and 4. The distance of author fragment was 15mm. Using the formula obtained from the graph figure2 (distance = 43.95 – 11.62 * log MW) the fragments size was calculated
|
Formula used |
Calculations |
|
(distance = 43.95 – 11.62 * log MW) Y= C – mx Y = distance C = 43.95 m = – 11.62 log MW = unknown
|
Y = 43.95 – 11.62 log MW 15 = 43.95 – 11.62 log MW 15 – 43.95 = (- 11.62) log MW – 28.95 = (- 11.62) log MW Log MW = (- 28.95) / (- 11.62) Log MW = 2.491bp
|
|
So the inti – log for the log MW = 310bp So 289bp for both digested and undigested.
|
|
The BLAST analysis was then performed to identify the primer binding region in the gene in DNA sequence of Homo sapiens (human).The analysis is as shown below:
CCTTTCTGCA CTGGGTGGCA ACCAGGTCTT TAGATTAGCC AACTAGAGAA GAGAAGTAGAATAGCCAATT AGAGAAGTGA CATC
ATGTTGACTCTAACTCGCAT CCGCACTGTG TCCTATGAAG TCAGGAGTAC ATTTCTGTTCATTTCAGTCCTGGAGTTTGC AGTGGGGTTT CTGACCAATG CCTTCGTTTT CTTGGTGAATTTTTGGGATG TAGTGAAGAG GCAGGCACTG AGCAACAGTG ATTGTGTGCT GCTGTGTCTCAGCATCAGCC GGCTTTTCCT GCATGGACTG CTGTTCCTGA GTGCTATCCA GCTTACCCACTTCCAGAAGT TGAGTGAACC ACTGAACCAC AGCTACCAAG CCATCATCAT GCTATGGATGATTGCAAACC AAGCCAACCT CTGGCTTGCT GCCTGCCTCA GCCTGCTTTA CTGCTCCAAGCTCATCCGTT TCTCTCACAC CTTCCTGATC TGCTTGGCAA GCTGGGTCTC CAGGAAGATCTCCCAGATGC TCCTGGGTAT TATTCTTTGC TCCTGCATCT GCACTGTCCT CTGTGTTTGGTGCTTTTTTA GCAGACCTCA CTTCACAGTC ACAACTGTGC TATTCATGAA TAACAATACAAGGCTCAACT GGCAGATTAA AGATCTCAAT TTATTTTATT CCTTTCTCTT CTGCTATCTGTGGTCTGTGC CTCCTTTCCT ATTGTTTCTG GTTTCTTCTG GGATGCTGAC TGTCTCCCTGGGAAGGCACA TGAGGACAAT GAAGGTCTAT ACCAGAAACT CTCGTGACCC CAGCCTGGAGGCCCACATTA AAGCCCTCAA GTCTCTTGTC TCCTTTTTCT GCTTCTTTGT GATATCATCCTGTGCTGCCT TCATCTCTGT GCCCCTACTG ATTCTGTGGC GCGACAAAAT AGGGGTGATGGTTTGTGTTG GGATAATGGC AGCTTGTCCC TCTGGGCATG CAGCCATCCT GATCTCAGGCAATGCCAAGT TGAGGAGAGC TGTGATGACC ATTCTGCTCT GGGCTCAGAG CAGCCTGAAGGTAAGAGCCG ACCACAAGGC AGATTCCCGG ACACTGTGCT GA
GAATGGAC ATGAAATGAG CTCTTCATTA ATACGCCTGT GAGTCTTCAT AAATATGCC
Figure 3: shows the DNA sequences by using the primer sequence & BLAST programme, the actual DNA sequence of gene amplification was obtained from PubMed (http://blast.ncbi.nlm.nih.gov/Blast.cgi
The figure illustrating the full length which is 1143 bp (base pair). Define as homo sapiens chromosome 7. Also it has been indicated the following:
PTC Forward 1 AACTGGCAGAATAAAGATCTCAATTTAT
PTC Reverse 2 AACACAAACCATCACCCCTATTTT
COMPLEMENTARY OF REV.PRIMERAAAATAGGGGTGATGGTTTGTGTT
START CODON ATG
STOP CODON TGA
Restriction enzyme with a mutation that located at 785 nucleotide where the C with taster to T in non-taster
There 3 mutations located at 145, 785, and 886
|
Gene details: |
LOCUS NC_000007 1143 bp DNA linear CON 06-JUN-2016 |
DEFINITION Homo sapiens chromosome 7, GRCh38.p7 Primary Assembly. |
ACCESSION NC_000007 REGION: complement(141972631..141973773) |
VERSION NC_000007.14
|
DBLINK BioProject: PRJNA168
Assembly : GCF 000001405.33
|
KEYWORDS RefSeq.
|
SOURCE Homo sapiens (human)
|
Figure 4: shows the detail of TAS2R38 gene obtained from NCBI
|
mRNA details: |
|
LOCUS NM_176817 1143 bp mRNA linear PRI 06-FEB-2018 |
|
DEFINITION Homo sapiens taste 2 receptor member 38 (TAS2R38), mRNA. |
|
ACCESSION NM_176817 |
|
VERSION NM_176817.4 |
|
KEYWORDS RefSeq. |
|
SOURCE Homo sapiens (human) |
Figure 5: shows the detail of TAS2R38 mRNA obtained from NCBI
|
Protein details: |
LOCUS NP_789787 333 aa linear PRI 06-FEB-2018
|
DEFINITION taste receptor type 2 member 38 [Homo sapiens].
|
ACCESSION NP_789787
|
VERSION NP_789787.4
|
DBSOURCE REFSEQ: accession NM 176817.4
|
KEYWORDS RefSeq.
|
SOURCE Homo sapiens (human)
|
Figure 6: shows the detail of TAS2R38 protein obtained from NCBI
Table 3: shows the percentage and the frequency of 75 students who are either a homozygous or heterozygous
|
Genotype |
taster genotype (+/+) (C/C) (TT) |
mild tasters (+/-) (C/T) (Tt) |
non tasters (-/-) (T/T) (tt) |
|
Number of students |
15 |
34 |
26 |
|
Frequency |
0.2 |
0.45 |
0.35 |
|
Frequency as percentage |
20% |
45% |
35% |
Table 4: Shows calculate the percentage and the allele frequency of student’s class
|
Allele type |
(-) (T) |
(+) (C) |
|
Number (150) |
86 |
64 |
|
Allele frequency & percentage |
0.57
(57%) |
0.43
(43%) |
Table 5: Shows the percentage and the frequency of 226 persons who are either a homozygous or heterozygous (European cohort)
|
Genotype |
taster genotype (+/+) (C/C) (TT) |
mild tasters (+/-)(C/T) (Tt) |
non tasters (-/-) (T/T) (tt) |
|
Number of persons |
44 |
106 |
76 |
|
Frequency |
0.195 |
0.469 |
0.336 |
|
Frequency as percentage |
20% |
47% |
33% |
Table 6: Shows the percentage and the allele frequency of European people
|
Allele type |
(-) (T) |
(+) (C) |
|
Number (452) |
258 |
194 |
|
Allele frequency & Percentage |
0.570
(57%) |
0.429
(43%) |
Table 7: Illustrates the percentage and the frequency of 224 persons who are either a homozygous or heterozygous (sub-Sahara cohort)
|
Genotype |
taster genotype (+/+) (C/C) (TT) |
mild tasters (+/-) (+/-)(C/T) (Tt) |
non tasters (-/-) (T/T) (tt) |
|
Number of persons |
100 |
106 |
18 |
|
Frequency |
0.446 |
0.473 |
0.08 |
|
Frequency as percentage |
45% |
47% |
8% |
Table 8: Shows the percentage and the allele frequency of sub-Sahara people
|
Allele type |
(-) (T) |
(+) (C) |
|
Number (448) |
142 |
306 |
|
Allele frequency & percentage |
0.316
(32%) |
0.683
(68%) |
Chi- squared test:
Chi – squared test can be obtained for checking if the two set of comparisons have a significant differences on their data or no by looking at the P-Value. If the P-Value is, lower than 0.05 that mean the two values are dependant, which reflect the reality of the data and vice versa.
|
Chi-Square Test: students, Sub-Sahara Expected counts are printed below observed counts Chi-Square contributions are printed below expected counts
|
|||
|
|
Students |
Sub-Sahara |
Total |
|
T |
86 57.19 14.513
|
142 170.81 4.859 |
228 |
|
C |
63 92.81 8.943
|
306 277.19 4.994 |
370 |
|
Total |
150
|
448 |
598 |
|
Chi-Sq = 31.309, DF = 1, P-Value = 0.000
|
|||
Figure 7: shows the result of chi-square for student and sub-Saharan. From chi square test we can note that all results were non-significant which indicate no differences between Sub-Saharan and class data results in the ability to taste PTC.
|
Chi-Square Test: students, European Expected counts are printed below observed counts Chi-Square contributions are printed below expected counts
|
|||
|
|
Students |
European |
Total |
|
T |
86 85.71 0.0009
|
258 258.29 0.0003 |
344 |
|
C |
64 64.29 0.0012
|
194 193.71 0.0004 |
258 |
|
Total |
150
|
452 |
602 |
|
Chi-Sq = 0.003, DF = 1, P-Value = 0.957
|
|||
Figure 8: shows the result of chi-square for student and European
As it is seen the result of P- value is higher than 0.05 that means there is a significant variety in the data between the two cohort which indicate there is a big differences in the ability of tasting. And that could be relating to the differences of the population specifically in the cohort of the class where as it known the class having some student who are an international students from different countries and different continent such as Asia, Africa, and European
5. Discussion (1000 words) (30%)
Discuss if there is any differences between the data
If there is any clinical relevance it should be discussed and talk about studies thats been used
evaluate individual/class experimental results, relate data to published data, Chi squared analysis, clinical relevance (diet, diseases, health other taste SNP’s) & conclusion.
6. References/Layout ( 20 Vancouver style) (5 %)
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