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තාරකා විද්යාව සඳහා DNA තාක්ෂණය
DNA (Deoxyribose Nucleic Acid) යනු ඔබ අප සැම කවුරුත් දන්නා බොහෝ ජීවී සෛල වල බහුලව හමුවන ජෛව බහුඅවයවිකයෙකි. ඒවායේ නියුක්ලියෝටයිඩ දාම වල ජීවියෙකුගේ ප්රවේණික තොරතුරු ගබඩා කර තබනු ලබන අතර අවශ්ය අවස්ථාවලදී ඒවා ප්රකාශ කිරීම හා පරම්පරාවෙන් පරම්පරාවට සම්ප්රේෂණය කිරීම සිදුකරනු ලබයි. එලෙසම මෙම DNA අණුවල සිදුවන විකෘති ජීවීන් පරිණාමය වීමට හේතු වේ. මෙම DNA අණුවල ව්යුහය, ක්රියාකාරීත්වය හා ඵලදායි භාවිතයන් පිළිබඳ අධ්යයනය කිරීම DNA තාක්ෂණයේ දී සිදු වේ.

තාරකා විද්යාව සඳහා DNA තාක්ෂණය වැදගත් වන්නේ කෙසේද?
මීට වසර බිලියන 3.5 කට පමණ පෙර පෘථිවියේ ජීවය ආරම්භ විය. එකල සිට අද දක්වා ජීවීන්ගේ සිදුවූ පරිණාමයන් සහ විකිරණයන් පිළිබඳ අධ්යනය තාරකා විද්යාවේ දී සිදුකරනු ලබයි. මෙලෙස පරිණාමය වීමේ දී ජීවීන්ගේ සිදු වූ අණුක මට්ටමේ වෙනස්වීම් සහ පරිණාමය සිදු වූ ආකාරය පිළිබඳව අධ්යනය සඳහා DNA තාක්ෂණය වැදගත් වේ.
ජීවියෙකුගේ අභ්යන්තර හා බාහිර පරිසරයේ ඇති භෞතික හා රසායනික සාධක වල බලපෑම මගින් DNA අණුවල විකෘති (Mutation) ඇති විය හැක. (ජීවියෙකුගේ ගෙනෝමයට අයත් නියුක්ලියෝටයිඩ අනුක්රමයක සිදුවන වෙනස්වීම් විකෘති ලෙස හැඳින්වේ.) මෙලෙස විකෘති ඇති වීමට හේතු වන සාධක අධ්යනය කිරීම මගින් පෘථිවියේදි හා පෘථිවියෙන් පිටත අභ්යවකාශයේදී ජාන වලට සිදුවන බලපෑම් පාලනය කිරීමට පූර්වෝපායන් සැලසුම් කිරීමට හැකි වනු ඇත.
ප්රතිසංයෝජිත DNA තාක්ෂණය (Recombinant DNA Technology) භාවිතයෙන් ප්රවේණිකව විකරණය කරන ලද ජීවීන් (GMO – Genetically Modified Organism) නිර්මාණය කිරීම වර්තමානයේ විද්යාඥයන් අතර බහුල හා ජනප්රිය ක්රියාන්විතයකි. මෙම ප්රවේණිකව විකරණය කරන ලද ජීවීන් භාවිතයෙන් Space Farming වැනි පෘථිවියෙන් පිටත අභ්යවකාශයේ හෝ වෙනත් ග්රහලෝකයක ජීවය ව්යාප්ත කිරීමෙහි පර්යේෂණ හා අනෙකුත් තාරකා විද්යාත්මක පර්යේෂණ කාර්යක්ශම කරගත හැක .
විශේෂයෙන් විශේෂයට මෙන්ම ජීවියාගෙන් ජීවියාට ද DNA අනුක්රමය එකිනෙකට වෙනස් වේ. මෙම DNA අනුක්රම අධ්යයනය කිරීම මඟින් අදාළ ජීවියාගේ හෝ අදාළ ජීවී විශේෂයේ සුවිශේෂී අනන්ය ලක්ෂණ හඳුනාගත හැකි වන අතර නව ජීවී විශේෂ (??? පිටසක්වල ජීවීන්/Aliens ???) හඳුනා ගැනීමට ද හැකි වනු ඇත.
DNA පිළිබඳ අධ්යයනයේදි ප්රතිසංයෝජිත DNA තාක්ෂණය (Recombinant DNA Technology) ඉතා වැදගත් ශිල්ප ක්රමයක් බවට පත්ව ඇත.
ප්රතිසංයෝජිත DNA තාක්ෂණයේ දී යොදා ගන්නා ශිල්ප ක්රම හා මෙවලම් කිහිපයක් පිළිබඳව අපි දැන් සලකා බලමු.
- DNA විසංගමනය
- ඇගරොස් ජෙල විද්යුතාගමනය
- DNA ඒෂණ සහ දෙමුහුම්කරණය
- ජාන තාක්ෂණයේ දී DNA සමඟ ප්රතික්රියා කරන එන්සයිම
DNA විසංගමනය (Isolation of DNA)
ජාන තාක්ෂණයේ පළමු පියවර දායක සෛලයක DNA ගෙනෝමයෙන් අදාල DNA අණුව නිස්සාරණය කර ගැනීමයි. මෙම ක්රියාවලිය DNA විසංගමනය ලෙස හැඳින්වේ.
DNA සූන්යෂ්ටික සෛලවල න්යෂ්ටිය තුල ද ප්රාග්න්යෂ්ටික සෛල වල සෛල තුළ ද සංචිත වී ඇත. එබැවින් DNA විසංගමනයේ පළමු පියවර වන්නේ අදාළ ඉලක්ක DNA අනුක්රමය සහිත DNA ගෙනොමය අදාල සෛලයෙන් පිටතට ගැනීමයි. ඒ සඳහා සෛල ප්ලාස්ම පටලය ද ශාක සෛලයක නම් සෛල බිත්තිය ද බිඳ දැමීම සිදු කළ යුතුයි. මෙය සමජාතීකරණය (Homogenization) හෝ ඇඹරීම වැනි යාන්ත්රික ක්රම මගින් මෙන්ම රසායනික ව ද සිදු කළ හැක. රසායනික ජීරණය යෙදී ලයිසොසයිම් වැනි එන්සයිම මගින් මෙන්ම මද්යසාර, ඊතර්, ක්ලෝරෆෝම් වැනි කාබනික රසායන සංයෝග මගින් ද සෛල බිත්තිය බිඳ දැමිය හැක .
ඇතැම් සෛල වල සෛල ප්ලාස්මයේ Deoxyribonuclease වැනි DNA ජීර්ණය කරන එන්සයිම ඇත. සමජාතීකරණය යෙදී න්යෂ්ටි පටලය බිඳ වැටීම නිසා DNA මෙම එන්සයිම සමඟ ගැටි ජීරණය විය හැක. එය සිදුවීම වැළැක්වීම සඳහා මෙම එන්සයිම උත්ප්රේරක ක්රියාවලියේ දී සක්රියකාරක ලෙස දායක වන ලෝහ අයන ඉවත් කිරීම සිදු කරයි. එම ලෝහ අයන ඉවත් කිරීම සඳහා නඛරියකාරක (Chelating Agents) භාවිතා කරයි. සුලබවම භාවිතා වන නඛරීයකාරකයක් ලෙස Ethylenediaminetetraacetic Acid (EDTA) දැක්විය හැක.
සෛල තුලදි DNA පවතින්නේ histone protein සමග බැඳී නියුක්ලියොප්රෝටික සංකීර්ණයක් ලෙස ය. DNA අධ්යනය කිරීම සඳහා ඒවා සමඟ බැඳී ඇති ප්රෝටීන ඉවත් කළ යුතුය. ඒ සඳහා Sodium dodecyl sulfate (SDS), Phenol හෝ ප්රොටියෝලිටික එන්සයිම භාවිතා කළ හැක. සෛලයක පවතින අනෙකුත් සියළුම අනු කොටස් (ලිපිඩ, RNA, cellulose) DNA සඳහා අපවිත්රකාරක වන අතර ඒවා ඉවත් කර ගත යුතුය.
DNA විසංගමනය අවසානයේදී DNA අණු C ශීත එතනෝල් මගින් අවක්ෂේප කරගත යුතුය. මෙම අවක්ෂේපය නැවත ස්වාරක්ෂක ද්රාවණයක දිය කරනු ලැබේ. මෙලෙස නිස්සාරණය කරගත් DNA අණු ඉදිරි පර්යේෂණ කටයුතු සඳහා භාවිතා කරනු ලැබේ
ඇගරොස් ජෙල විද්යුතාගමනය
විශාල ආරෝපිත අණු විද්යුත් ක්ෂේත්රයක දී ඒවායේ සචලතාවයට අනුව වෙන් කර ගැනීමේ ශිල්ප ක්රමය විද්යුතාගමනය ලෙස හැඳින්වේ. එහිදී අණුවල විශාලත්වය (කුඩා අණු විශාල අණු වලට සාපේක්ෂව වේගයෙන් ගමන් කරයි) හා ශුද්ධ ධ්රැවීයතාව මත අණු වෙන් කර ගැනීම සිදු වේ. DNA වෙන්කරගැනීමට බහුලව යොදා ගන්නා ශිල්ප ක්රමය වන්නේ ඇගරොස් ජෙල විද්යුතාගමනය යි.


එහිදී විද්යුතගමන උපකරණයේ පූරකය ලෙස ඇගරොස් නම් එගාර් විශේෂයක් භාවිතාවේ. පූරකය දෙපස ඇනෝඩයක් සහ කැතෝඩයක් තබා පූරකය තුළ සෘණ ආරෝපිත DNA අණු තැන්පත් කරනු ලබයි. විද්යුත් ක්ෂේත්රය ක්රියාත්මක කළ විට දිග අනුව DNA අණු වෙන් වීම සිදු වේ.

වෙන් කරන ලද DNA අණු එතිඩියම් බ්රෝමයිඩ් හෝ N-719 (කාබනික වර්ණකයකි) මගින් වර්ණ ගැන්විය හැක. එතිඩියම් බ්රෝමයිඩ් මගින් වර්ණ ගන්වන ලද DNA අණු UV කිරණ වලට නිරාවරණය කිරීමෙන් හඳුනාගත හැක.

DNA ඒෂණ සහ දෙමුහුම්කරණය (DNA Probes and Hybridization)
දෙමුහුම්කරණය මගින් අනුපූරක නියුක්ලියෝටයිඩ අනුක්රමණයක් හඳුනාගැනීම සඳහා භාවිතා වන තනි දාම, සලකුණු කරන ලද DNA ඛණ්ඩයක් ඒෂනයක් ලෙස හැඳින්වේ .ඒෂනයට විකිරණශීලී සමස්ථානිකයක් හෝ ප්රතිදීප්ත අණුවක් සම්බන්ධ කිරීමෙන් සලකුණු කිරීම සිදුකරනු ලබයි. ඒෂනයට අනුපූරක DNA හෝ RNA සමග දෙමුහුම්කරණය විය හැක.
දෙමුහුම්කරණයට පෙර DNA අණුවේ දාම දෙක වෙන් කර ගත යුතුය. දුස්ස්වභාවිකරණය මගින් DNA පට 2 වෙන් කරගනු ලැබේ. දුස්ස්වභාවිකරණය කරන ලද DNA පට නයිට්රෝසෙලියුලොස් හෝ නයිලෝන් පෙරහන් පටලයකට මාරු කළ යුතුය. මෙම ක්රියාවලිය Southern Blotting ලෙස හැඳින්වේ. අනතුරුව පටල මතට සලකුණු කරන ලද ඒෂණ එකතුකර ද්විපට සෑදීමට ඉඩ හරි.
පටලය සේදූ විට DNA අණුවට බැඳුණු ඒෂනය හැර අනෙකුත් ඒෂන සියල්ල ඉවත් වේ .විකිරණශීලී සමස්ථානිකයක් මගින් සලකුණු කරන ලද ඒෂණ ස්වයං විකිරණ රේඛනය මගින් හඳුනාගත හැක. ප්රතිදීප්ත අණුවකින් සලකුණු කරන ලද DNA UV කිරණ මගින් හඳුනාගත හැක.
ජාන තාක්ෂණයේ දී DNA සමඟ ප්රතික්රියා කරන එන්සයිම
ප්රතිසංයෝජිත DNA අණුවක් සෑදීමේදී DNA අණු කැපීම DNA අණු කොටස් එකිනෙක සම්බන්ධ කිරීම DNA දාම වෙන්කිරීම වැනි ක්රියාවන් රැසක් සිදුකළ යුතුය .එම ක්රියාවලින් කාර්යක්ෂමව හා ඵලදායීව සිදු කර ගැනීම සඳහා ජෛව රසායනික ප්රතික්රියාවන් උත්ප්රේරණය කරන එන්සයිම ජාන තාක්ෂණයේ දී භාවිතා වේ.
විශිෂ්ට DNA අනුක්රමයක් හඳුනා ගෙන ඒවායේ කැපිය යුතු ස්ථානවලින් හෝ ඒ අසලින් කැපුම් සිදුකිරීම උත්ප්රේරණය කරන එන්සයිමයක් ලෙස Restriction endonuclease ගත හැක. මෙම කැපුම් යෙදෙන ස්ථාන සීමාකාරී ස්ථාන හෝ ජේදන ස්ථාන ලෙස හැඳින්වේ.
විවිධ ප්රභව වලින් ලබාගත් කැපූ DNA කොටස් Phosphodiester බන්ධන මගින් එකිනෙක සම්බන්ධ කර ප්රතිසංයෝජිත DNA ලබාගැනීම DNA Ligase එන්සයිමය මගින් උත්ප්රේරණය කරනු ලැබේ. T4DNA Ligase ජාන තාක්ෂණයේ දී බහුලව යොදා ගන්නා ligase වර්ගයකි.
DNA අච්චු දාමයට අනුපූරක නියුක්ලියෝටයිඩ එක්කර නව DNA දාමයක් සෑදීම DNA polymerase එන්සයිමය මගින් උත්ප්රේරණය කරනු ලැබේ.බහුලවම භාවිතා කරන පොලිමරේස් වර්ගය වන්නේ Taq DNA polymerase.
මීට අමතරව ජාන තාක්ෂණයේ දී Alkaline Phosphatase, Reverse Transcriptase, Kinase, Phosphatase, Terminal Deoxynucleotidyl Transferase වැනි එන්සයිම රාශියක් භාවිතා කරනු ලැබේ .
ඉහත සඳහන් කල ශිල්ප ක්රම වලට අමතරව DNA ආශ්රිත තවත් ශිල්ප ක්රම රාශියක් පවතී. ඒවා තාරකා විද්යාවේ අභිවෘද්ධිය සඳහා නන් අයුරින් යොදා ගත හැක.
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