Chapter 3: basic principles of molecular

marker (DNA marker) and some

biotechnology techniques  

Part  6:  Introduce  some  DNA  markers    

Part 7: DNA, PCR, Electrophoresis methods
Part  6:  Introduce  some  DNA  markers    
 DNA Markers
1. Restriction fragment length polymorphisms (RFLPs)
2. Amplified fragment length polymorphisms (AFLPs)
3. Random amplified polymorphic DNA (RAPDs)
4. Simple sequence repeats (Microsatellites - SSRs)
5. Single nucleotide (SNPs)
6. VNTR
7. Sequencing
RFLP marker　　　　　　　　 RAPD marker 　　　　 Microsatellite marker
 Restric7on  Fragment  Length  
Polymorphism  (RFLP)

Enzymes  cut  DNA  at  specific  sequences  
Restric7on  sites  are  oEen  palindromes:    
6-­‐cuGer  GAATTC  4-­‐cuGer            TCGA  
                         CTTAAG        AGCT  
 Marker RFLP
Restriction Fragment Length Polymorphisms
Restriction
site

Single nu
S7cky  ends  
DNA separation by gel electrophoresis

- Small DNA fragments move further through the gel

than large fragments
DNA separation by gel electrophoresis
The hold contains DNA sample

− �
+�

Gel�

Negative (-) Positive (+)

DNA Matrix
Marker
 B

Restriction fragment length polymorphism (RFLP)

A B A B

B
Can  be  used  for  species  or  popula7on  
iden7fica7on  

•  Human  mt  DNA  has  2  EcoR1  restric7on  

sites  
•  Honey  bee  mt  DNA  has  5  restric7on  sites  

Q: How many bands would you see on a gel after digesting

human and honey bee mtDNA with the EcoR1 restriction enzyme?
Hint: mtDNA is circular in both humans and honey bees.!
 
 Can  be  used  for  analysis  of  
relatedness  

“Ladder”  
Using  RFLP  polymorphism  to  study  popula7on  
gene7c  structure  and  evolu7on  
Advantages:  variants  are  co-­‐dominant;  
measures  varia7on  at  the  level  of  DNA  
sequence,  not  protein  sequence.  
Disadvantages:  labor  intensive;  requires  
rela7vely  large  amounts  of  DNA  
 
 PCR  based  methods:  
don’t  need  much  DNA  

•  RAPD:  randomly  amplified  polymorphic  

DNA  
•  AFLP:  amplified  fragment  length  
polymorphism  
•  VNTR:  variable  number  tandem  repeats;  
including  microsatellites  
PCR:  polymerase  chain  reac7on  

3’  
5’  
5’  
3’  
RAPD:  randomly  amplified  
polymorphic  DNA  

Size sorted!
 Marker chỉ thị RADP
Marker Random Amplified Polymorphic

Hình: Marker RAPD

 RAPDs  
Advantages:  fast,  rela7vely  inexpensive,  highly  
variable.  
Disadvantages:  markers  are  dominant.  Presence  
of  a  band  could  mean  the  individual  is  either  
heterozygous  or  homozygous  for  the  
sequence-­‐-­‐can’t  tell  which.  Data  analysis  
more  complicated.  
RAPD  Analysis   Ques7ons:  
1.  Is  the  locus  represented  by  
band  “B”  polymorphic?  Band  
A?  
B  
2.  Is  individual  232  a  
homozygote  or  heterozygote  
for  alleles  represented  by  
band  “B”?    What  about  
individual  236?  
3.  Does  band  “B”  represent  a  
longer  or  shorter  DNA  
fragment  than  band  “A”.    
AFLP:  amplified  fragment  length  
polymorphism  

Digestion of DNA with two

enzymes
Ligation of adapters to
fragment ends
Primers complementary to
adapters and to 3’ region of
some of the fragments
 Marker

Marker chỉ thị AFLP

(Amplified Fragment
Length Polymorphisms)
AFLPs  
 AFLPs  
Advantages:  fast,  rela7vely  inexpensive,  highly  
variable.  
Disadvantages:  markers  are  dominant.  Presence  
of  a  band  could  mean  the  individual  is  either  
heterozygous  or  homozygous  for  the  
sequence-­‐-­‐can’t  tell  which.  
 RAPDs  and  AFLPs  
Good  for  dis7nguishing  between  popula7ons  
OEen  used  for  trait  mapping  studies  because  
they  are  variable  between  the  popula7ons  
that  are  crossed  
 VNTR:  variable  number  tandem  
repeats  

•  Non-­‐coding  regions  
•  Several  to  many  copies  of  the  same  
sequence  
•  Large  amount  of  varia7on  among  
individuals  in  the  number  of  copies  
 Microsatellites  
•  Not  a  7ny  orbi7ng  space  craE  
•  Most  useful  VNTRs  
•  2,  3,  or  4  base-­‐pair  repeats  
•  A  few  to  100  tandem  copies  
•  Highly  variable  
•  Many  different  microsatellite  loci  (1000s)  in  
any  species  
 Microsatellites  
•  Design  primers  to  flanking  regions  
Microsatellite  Gels  
 Microsatellites  

Advantages:  highly  variable,  fast  evolving,  co-­‐

domininant  
Rela7vely  expensive  and  7me  consuming  to  
develop  
Microsatellite marker

Microsatellite marker = SSR (simple sequence repeat) marker

Repeat unit - simple sequence (mono-, di-, tri-, tetranucleotides)
ex. A AAAAA……AAAAA
GA GAGAGAGA……GAGAGAGA
ATT ATTATTATT……ATTATTATT

SSLP (Simple sequence length polymorphism)

---GAGAGAGAGAGAGAGAGAGAGAGA--- (GA)12

---GAGAGAGAGAGAGAGAGAGAGAGA--- (GA)12 0
---GAGAGAGAGAGAGAGAGAGAGAGAGAGAGA--- (GA)15 +6
---GAGAGAGAGAGAGAGAGAGA--- (GA)10 -4
Hình: Đa hình đoạn lặp lại
 Microsatellites  
Used  for  within-­‐
popula7on  studies;  
not  as  much  for  
between-­‐popula7on  
studies  b/c  they  
evolve  too  fast  
Paternity  analysis  and  
other  studies  of  
kinship  
 Microsatellites  
Ques7ons:  
1.  Is  the  locus  represented  
by  the  bands  at  the  
arrow  polymorphic?  
2.  If  it  is  polymorphic,  how  
many  individuals  are  
heterozygous?  
3.  How  many  individuals  
are  homozygous  for  the  
“short”  allele?  
 
Sequencing  
 Sequencing  

OEen  used  for  phylogene7cs  (especially  

sequences  of  mitochondrial  genes).  
Also  used  for  studies  of  molecular  evolu7on  
(e.g.,  compare  rates  of  synonymous  vs.  
non-­‐synonymous  subs7tu7on)  
Sequencing  

Q: What’s the DNA sequence?!

Molecular marker
Dựa trên sự biến đổi (đột biến) về mức độ di truyền DNA

Base substitution�
�
A ----------GAGTCGAATTCGATTCTG----------�
�
B ----------GAGTCGAGTTCGATTCTG----------�
�
Insertion / deletion�
�
A ----------GAGTCGAATTCGATTCTG----------�
�
B ----------GAGTCGGATTCTG----------�
�
Multiplication�
�
A ----------□□□----------�
�
B ----------□□□□□□□----------�
Part 7: DNA, PCR, Electrophoresis methods
DNA extraction   Where is DNA in plant cell ?
DNA extraction
- How to work with DNA ? (DNA is very small, it
can not see and smell)

- But it is very easy to amplify from 1 copy to many

copies using PCR method (Poly Chain Reaction)
 Target region
between two primers

Template DNA

Denature Denature
Extension Extension 2 molecules 4 molecules
Annealing Annealing

1st cycle 2nd cycle 3rd cycle 4th cycle

DNA amplification by PCR

Hình: Cơ chế biến tính DNA
 PCR (Polymerase Chain Reaction)

Primer
Primer

Synthesis
Basis elements use for DNA amplification
(PCR method)
-  Pairs of primer
-  dNTP
-  Enzyme DNA polymerase
-  Buffer và Mg2+
-  DNA template
DNA separation by gel electrophoresis

- Small DNA fragments move further through the gel

than large fragments