Lecture

 5  
Basics  of  DNA  Cloning  

Cloning  is  making  of  iden/cal  copies.  DNA  cloning  is  process  of  making  several  iden/cal  
copy  of  a  gene  or  gene  fragment.  DNA  fragment  from  an  organism  is  cleaved  or  amplified  
and  inserted  in  a  DNA  carrier  called  vector.    
Cloning   is   a   natural   process   in   biology   where   gene/cally   iden/cal   individuals   are   produced   by   asexually  
reproducing   organisms   such   as   bacteria,   insects   or   plants.   In   biotechnology,   the   process   of   producing  
mul/ple  iden/cal  copies  of  DNA  fragments  (molecular  cloning),  cells  (cell  cloning),  or  organisms  is  referred  
to  as  cloning.  A  clone  has  an  exact  gene/c  imprint  as  that  of  the  original  cell,  /ssue  or  organism.  

There  are  different  types  of  cloning    

In  Biotechnology  the  gene  is  the  cornerstone  of  most  molecular  biology  studies.  The  study  of  genes  can  be  
facilitated  by  isola/on  and  amplifica/on  of  gene  of  interest.  Cloning  is  one  method  used  for  isola/on  and  
amplifica/on  of  gene  of  interest.    
 Illustra/on  of  possible  reading  frames:  
AGG·∙TGA·∙CAC·∙CGC·∙AAG·∙CCT·∙TAT·∙ATT·∙AGC  
A·∙GGT·∙GAC·∙ACC·∙GCA·∙AGC·∙CTT·∙ATA·∙TTA·∙GC  
AG·∙GTG·∙ACA·∙CCG·∙CAA·∙GCC·∙TTA·∙TAT·∙TAG·∙C  

An  open  reading  frame  (ORF)  is  a  reading  frame  that  has  the  poten/al  to  be  transcribed  
into  RNA  and  translated  into  protein.  It  requires  a  con/nuous  sequence  of  DNA  from  a  
start  codon,  through  a  subsequent  region  which  usually  has  a  length  that  is  a  mul/ple  of  
3  nucleo/des,  to  a  stop  codon  in  the  same  reading  frame  
Dr.  Ban  Oday  AbdulsaRar  
Dr.  Ban  Oday  AbdulsaRar  
 Steps  of  cloning  
Select gene(CTXM-15)

PCR amplification

pTriEx1.1

In-Fusion

Colony PCR Sequencing

Transformation

Double digestion
Cloning  steps:  
The  first  step  in  cloning  is  to  prepare  large  amount  of  the  vector  and  chromosomal  DNAs  
all   vectors   are   self   replica/ng   in   the   cell,   they   contain   a   number   of   unique   restric/on   enzyme  
cleaving   sites   that   are   present   only   once   in   the   vector,   they   carry   the   selectable   marker   gene  
which  is  useful  in  selec/on  of  clone  (usually  an  an/bio/c  resistance  gene  that  is  absent  in  the  
host   cell)   and,   they   can   be   very   easily   isolated   from   host   cell.   Depending   on   the   purpose   of  
cloning  there  are  many  vectors  available.  For  use  in  the  bacterial  host  E.  coli  system  a  greatest  
variety   of   cloning   vectors   have   been   developed.   Thus,   the   first   thing   in   cloning   that   a   molecular  
biologist  requires  is  to  grow  pure  culture  and  isolate  the  cloning  vector  from  the  cells.    
Choice  of  vector  is  dependent  on  insert  size  and  applica/on    
The  different  types  of  vectors  available  for  cloning  are  
plasmids,  bacteriophages,  bacterial  arAficial  chromosomes  
(BACs),  yeast  arAficial  chromosomes  (YACs)  and  mammalian  
arAficial  chromosomes  (MACs).    
Plasmids:  Plasmids  are  extra  chromosomal  circular  double  
stranded  DNA  replica/ng  elements  present  in  bacterial  cells.  
Plasmids  show  the  size  ranging  from  5.0  kb  to  400  kb.  
Plasmids  are  inserted  into  bacterial  calls  by  a  process  called  
transforma/on.  Plasmids  can  accommodate  an  insert  size  of  
upto  10  kb  DNA  fragment.  Generally  plasmid  vectors  carry  a  
marker  gene  which  is  mostly  a  gene  for  an/bio/c  resistance;  
thereby  making  any  cell  that  contains  the  plasmid  will  grow  in  
presence  of  the  selectable  corresponding  an/bio/c  supplied  
in  the  media.    
Bacteriophage:  The  viruses  that  infect  bacteria  are  called  bacteriophage.  These  are  intracellular  
obligate   parasites   that   mul/ply   inside   bacterial   cell   by   making   use   of   some   or   all   of   the   host  
enzymes.  Bacteriophages  have  a  very  high  significant  mechanism  for  delivering  its  genome  into  
bacterial  cell.  Hence  it  can  be  used  as  a  cloning  vector  to  deliver  larger  DNA  segments.    
Most  of  the  bacteriophage  genome  is  non-­‐essen/al  
and  can  be  replaced  with  foreign  DNA.  Using  bacteriophage    
as  a  vector,  a  DNA  fragment  of  size  up  to  20  kb    
can  be  transformed.  
 
 
 
 
Bacterial   ar/ficial   chromosomes   (BACs):   Bacterial   ar/ficial   chromosomes   (BACs)   are   simple  
plasmid  which  is  designed  to  clone  very  large  DNA  fragments  ranging  in  size  from  75  to  300  kb.  
BACs  are  basically  used  in  sequencing  the  genome  of  organisms  in  genome  projects  (example:  
BACs  were  used  in  human  genome  project).  Several  hundred  thousand  base  pair  DNA  fragments  
can  be  cloned  using  BACs.  
Yeast   ar/ficial   chromosomes   (YACs):   YACs   are   yeast   expression   vectors.   A   very   large   DNA  
fragments  whose  sizes  ranging  from  100  kb  to  3000  kb  can  be  cloned  using  YACs.  Mostly  YACs  
are   used   for   cloning   very   large   DNA   fragments   and   for   the   physical   mapping   of   complex  
genomes.    
Human   ar/ficial   chromosomes   (HACs):   Human   ar/ficial   chromosomes   (HACs)   or   mammalian  
ar/ficial  chromosomes  (MACs)  are  s/ll  under  development.  HACs  are  microchromosomes  that  
can  act  as  a  new  chromosome  in  a  popula/on  of  human  cells  
COSMID:It   is   a   cloning   vector   consis/ng   of   the  
phage   cos   site   inserted   into   a   plasmid   i.e.   simply  
plasmid   and   cos   sites.     It   is     used   to   clone   DNA  
fragments  upto  40  kb  in  size.    cos  site  is  one  of  the  
cohesive,  single  stranded  extensions  present  at  the  
ends   of   the   DNA   molecules   of   certain   strains   of  
lamda   phage.     The   main   reason   for   the  
development   of   the   cosmid   vector   is   to   irradiate  
the   disadvantages   of   plasmids   and   phage   vectors.    
Due   to   the   forma/on   of   cosmids   the   low   copy  
number  property  of  plasmids  overcome  because  of  
cos   sites   and   rolling   circle   replica/on   and   the   lysis  
of  culture  which  is  the  disadvantage  of  phage  virus  
overcome   because   the   vector   does   not   have  
sequence   for   the   func/onal   phage   produc/on.    
Aaer   the   recombinant   vector   mul/plied,   they   are  
packaged   into   virus   through   invitro   packaging  
method   because   of   the   cos   site   in   phage.    
Transforming   efficiency   increased   by   this   package  
method.  
Liga/on-­‐  joining  DNA  molecules  together  
The   ligase   enzyme,   called   Molecular   glue   are   pasted   these   DNA   fragments   into   the   carrying  
vector.  The  process  of  ligase  enzyme  for  joining  the  two  ends  of  DNA  strands  is  called  liga/on.  In  
this   process,   there   occurs   a   synthesis   of   phosphodiester   bond   between   the   3’hydroxyl   of   one  
nucleo/de  and  5’phosphate  of  another  
Liga/ng  an  insert  DNA  into  a  plasmid  requires  
complementary  ends  between  the  DNA  and  
the  plasmid  vector.    S/cky  ends  are  produced  
by  cueng  the  DNA  in  a  staggered  manner  
within  the  recogni/on  site  and  there  by  
produce  short  Single  stranded  DNA.  These  
ends  have  iden/cal  nucleo/de  sequence  and  
are  s/cky  because  they  can  bind  to  
complementary  tails  of  other  DNA  fragments  
cut  by  the  same  restric/on  enzyme.  Both  are  
useful  in  molecular  gene/cs  for  making  
recombinant  DNA  and  proteins.    
Different  parameters  affect  liga/ons  such  as  the  ra/o  of  insert  to  vector,  the  quality  and  type  of  
the  DNA  ends,  the  liga/on  temperature  and  the  DNA  concentra/on.  Each  of  these  factors  is  
necessary  for  a  successful  liga/on.      
Types  of  liga/on:  
i)  Linkers  
ii)  Adaptors  
iii)  Homopolymer  tailing  
 
i)  Linkers:  
Linker   molecules   are   used   to   ligate   the  
blunt   end   gene   of   interest   with   cohesive  
end  vectors.    They  are  normally  synthesized  
s e l f -­‐ c o m p l e m e n t a r y   d e c a m e r i c  
oligonucleo/edes,   which   contain   sites   for  
one   or   more   restric/on   endonucleases  
which  will  create  s/cky  ends.    The  linker  can  
be  ligated  to  both  ends  of  the  foreign  gene  
to   be   clones,   and   then   treated   with  
restric/on   endonuclease   to   produce   a  
s/cky   ended   fragment   which   can   be  
incorporated   into   a   vector   molecule   that  
has   been   cut   with   the   same   restric/on  
endonuclease.     Inser/on   by   means   of   the  
linkers   creates   restric/on   enzyme   target  
sites  at  each  end  of  the  foreign  gene  and  so  
enables  the  foreign  gene  to  be  excised  and  
recovered  aaer  cloning  and  amplifica/on  in  
the  host  bacterium.  
ii)  Adaptors:  When  linkers  added  to  link  at  the  end  of  blunt  end  of  gene  interest,  then  there  is  an  possibility  
of  joining  of  mul/ple  linkers  at  the  end.    This  makes    some  /me  larger  genes  and  waste  of  linker  molecules.      
This  problem  is  overcome  by  using  adapters.    Since  adapters  contain  only  one  end  suitable  for  joining  this  
prevents  mul/ple  coiling  of  adapters.    Adapter  is  a  synthe/c,  double  stranded  oligonucleo/de  used  to  aRach  
s/cky  ends  to  a  blunt  ended  molecule.  
 It  contain  normal  5'  and  3'  end  at  blunt  end  and  the  s/cky  end  of  adapter  molecule  is  modified  in  such  
manner  that  it  contain  OH  group  on  both  5'  and  3'  ends.    This  is  achieved  by  using  alkaline  phosphatases.    In  
contrast  to  linkers,  adapters  contain  preformed  s/cky  ends  and  joining  blunt  ends.    Because  of  lack  of  5'  
phosphate  group  on  s/cky  end  prevents  adapter    polymer  forma/on.  Aaer  the  adaptors  have  been  aRached  
the  abnormal  5'OH  terminus  is  converted  to  the  natural  5'P  form  by  treatment  with  the  enzyme  
polynucleo/de  kinase,  producing  s/cky  ended  fragment  that  can  be  inserted  into  an  appropriate  vector.  
Eventhough   adaptors   prevents   polymer  
forma/on,   it   does   not   prevents   self  
liga/on   or   recoiling   of   vectors   during  
r e c o m b i n a / o n   r e a c / o n .     T h i s  
disadvantages   nature   of   adaptors  
removed  by  using  homopolymer  tailing.  
iii)  Homopolymer  Tailing:  A  homopolymer  is  simply  a  polymer  in  which  all  the  subunits  are  the  
same.    Tailing  involves  using  the  enzyme  terminal  deoxynucleo/dyl  transferase,  to  add  a  series  
of  nucleo/des  on  to  the  3'-­‐OH  termini  of  a  double  stranded  DNA  molecule.    If  this  reac/on  is  
carried   out   in   the   presence   of   just   one   deoxynucleo/de,   then   a   homopolymer   tail   will   be  
produced.    In  this  method,  gene  of  interest  is  tailed  with  one  nucleo/de  and  vector  is  tailed  with  
an  complementary  base  and  when  they  are  combined  then  only  vector  recombined  with  gene  of  
interest.     In   this   case   recoiling   of   vectors   are   mostly   prevented   because   vector   does   not   contain  
complementary  ends.  
Applica/ons:-­‐  
Liga/on  of  cohesive  or  blunt-­‐ended  DNA  fragments  for  cloning  
Sealing  nicks  in  double-­‐stranded  DNA  
Liga/on  of  synthe/c  linkers  to  blunt-­‐ended  DNA  
Three  main  components  of  a  liga/on  reac/on  are:-­‐    
 
1)  Two  or  more  fragments  of  DNA  that  have  compa/ble  ends.  
 
2)  A  buffer  that  has  0.25-­‐1mM  ATP  to  provide  the  necessary  energy  for  the  reac/on.  
 
3)  The  T4  DNA  ligase.  
The  two  components  of  the  DNA  in  the  liga/on  reac/on  (vector  and  insert)  should  be  equimolar  
and  around  100μg/ml.  Usually,  one  wants  to  ligate  an  insert  DNA  molecule  into  a  plasmid,  ready  
for  bacterial  transforma/on.  Typically,  DNA  and  plasmid  vector  are  separately  cleaved  to  get  
complementary  ends,  then  both  are  added  to  a  liga/on  reac/on  to  be  circularised  by  DNA  
ligase.  If  the  ra/o  of  plasmid  backbone  to  insert  DNA  is  too  high  then  excess  'empty'  mono  and  
polymeric  plasmids  will  be  generated.  If  the  ra/o  is  too  low  results  in  an  excess  of  linear  and  
circular  homo-­‐  and  heteropolymers.The  ideal  ra/os  for  liga/ng  insert  to  vector  for  s/cky  end  
liga/ons  ranges  between  1:1  and  3:1,  where  as  for  blunt  ended  liga/ons,  the  insert  to  vector  
ra/o  should  be  at  least  10:1.  
Calcula/ng  Insert  Amount:-­‐  

hRps://nebiocalculator.neb.com/#!/liga/on