Clc genetics chapter 3

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 Restriction fragment length polymorphisms (RFLPs) Amplified fragment length polymorphisms (AFLPs) Random amplified polymorphic DNA (RAPDs) Simple sequence repeats (Microsatellites - SSRs) Single nucleotide (SNPs) VNTR 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� Positive (+) Negative (-) DNA Matrix 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 biến đổi (đột biến) mức độ di truyền DNA Base substitution� � A GAGTCGAATTCGATTCTG � B GAGTCGAGTTCGATTCTG � � � Insertion / deletion� � A GAGTCGAATTCGATTCTG � B GAGTCGGATTCTG � � � Multiplication� � □□□ � B □□□□□□□ � � A 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 copy to many copies using PCR method (Poly Chain Reaction) Target region between two primers Template DNA Denature Annealing Extension 1st cycle Denature Annealing molecules Extension 2nd cycle 3rd cycle DNA amplification by PCR molecules 4th cycle 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 Mg2+ -  DNA template DNA separation by gel electrophoresis - Small DNA fragments move further through the gel than large fragments