W11 DNA Sequence Analysis

Question 1

DNA Sequence analysis:

Answer 1

-DNA sequence analysis:
-Sanger dideoxynucleotide sequencing
-Pyrosequencing

DNA Amplification:
Polymerase Chain Reaction PCR

Question 2

Dideoxy DNA sequencing:

Answer 2

Processes used:
-Template
-DNA polymerase
-Primers
-Nucleotides

Question 3

Dideoxy nucleotides:

Answer 3

-Dideoxy refers to the nucleotides added to the DNA synthesis reaction
-The reaction contains both deoxy and di-deoxy nucleotide triphosphates

-These ddNTPs have an extra oxygen missing (compared to normal dNTPs)
-This modification means ddNTPs do not have a 3’-OH group
-ddNTPs cannot form a phosphodiester bond with any further nucleotide and the chain cannot be extended

Question 4

4 reactions in 1 tube:

Answer 4

-All 4 reactions occur in one tube
-ddG, ddC, ddA, ddT
-The ddNTPs can each be labelled with different fluroescent tags
-The extending chain can terminate at any nucleotide position if a ddNTP is incorporated
-All terminated chains are fluorescently labelled by one of four nucleotide specific colours
-If we know the size of DNA fragment and colour of label we know the DNA sequence

Question 5

Separation of fragments:

Answer 5

-Fragments are separated and sized in polyacrylamide gel held in fine capillaries (type of gel electrophoresis)
-Fragments are sorted by size, smallest exit capillary first, largest last
-Fragments differ in size by 1 nucleotide

Question 6

Dideoxy sequencing:

Answer 6

-As fragments pass end of capillary the fluorescent groups are excited by a laser
-Detection of 4 colour intensities is translated into sequence

Question 7

Pyrosequencing:

Answer 7

-No chain termination required
-dNTPs are added to the reaction mix one at a time and any not incorporated into the extending DNA strand is removed before addition of the next DNTP
-Incorporation of a dnTP into the extending DNA strand releases pyrophosphate
-Pyrophosphate is detected using an automated system

Question 8

Introduction to Polymerase chain reaction (PCR):

Answer 8

-PCR permits copying of DNA molecules in vitro
-Restriction digests of entire genomes produce large numbers of DNA fragments
-Amplification of a specific DNA sequence is possible using PCR

-Amplification of a specific DNA sequence is defined by a pair of DNA primers
-PCR synthesises copies of DNA in vitro using a similar process to DNA replication
-The reaction mixture contains:
-DNA polymerase enzyme (plus magnesium cofactor)
-dNTPs (nucleotides A,T,C,G)
-Oligonucleotide single stranded DNA Primers
-Template - the DNA to be copied

Question 9

PCR Continued:

Answer 9

-The reaction mixture is subjected to heating and cooling steps:
1) Denature DNA template - high temp (94-96)
2) Anneal primers to template (50-70)
3) Extend primers to synthesise a new copy strand (72)
-Due to high temperatures used, a thermostable enzyme is used, typically taq DNA polymerase
-The heating and cooling steps are repeated for several cycles
-The number of DNA copies increases exponentially each cycle, ie the number of copies doubles every cycle