Methods 2 (lecture 4) Flashcards
If you want to subclone a 2.5kb DNA fragment from genomic DNA that does not have suitable restriction sites (i.e., none flanking the insert or compatible with the multiple cloning site of the vector you need to use), how will you solve this problem and make the construct?
PCR amplify the fragment, design primers w/ extensions on the 5’ ends that contain restriction sites (~10nt) and match the DNA of interest on the 3’ ends (~20nt), use proofreading DNA polymerase for PCR, digest with appropriate restriction enzymes, ligate plasmid and fragment together, transform e. coli
What is a proofreading DNA polymerase
Proofreading DNA polymerase in PCR is an enzyme that can detect, remove, and replace incorrect bases in DNA during replication. This process is also known as 3’-5’ exonuclease activity.
Types of 1st gen DNA sequencing (2 types)
- Maxam-Gilbert chemical modification and cleavage (outdated)
- Sanger dideoxy chain-termination (relevant)
Types of next-gen or 2nd generation or massively parallel sequencing (4 types)
- 454 pyrosequencing, roche
- Illumina (Solexa) sequencing by synthesis
- SOLiD sequencing by ligation, life technologies (Applied Biosystems)
- Ion Torrent semiconductor, life technologies
Steps of Maxam-Gilbert sequencing
- 5’-ends of dsDNA are radioactively labeled by polynucleotide kinase and [g-32P]-ATP
- dsDNA is denatured in the presence of DMSO and heat, and ssDNA is purified*
- Labeled ssDNA is split into 4 reactions, chemically treated and cleaved after specific bases
- Obtained fragments are separated on a polyacrilamide gel and autoradiographed
Single-molecule or 3rd generation sequencing (3 types)
- Helicos Biosciences
- Single Molecule Real Time (SMRT), Pacific Biosciences
- Oxford Nanopore
In maxam-gilbert sequencing how is ssDNA purified?
centrifugation, one strand will have more purines (larger) one more pyrimidines (smaller)
In maxam-gilbert sequencing why is radiolabeled ssDNA split into 4 reactions
Different chemicals are used which break the bonds between different nucleotides, by running 4 seperate reactions on the same gel you can get base pair resolution of you DNA
Why do ssDNA need to be separated by size in the Maxam-Gilbert method?
I the both complementary strands are run on the gel you will not be able to tell which fragments belong to which strand as both will be bottom -> top or gel starting from labeled 5’ end
What is the name of the fluorescent nucleotide terminator utilized in Sanger sequencing?
fluorescently labeled dideoxynucleotide
What are the ingredients for sanger sequencing reaction
DNA polymerase I
Primer
dATP
dGTP
dCTP
dTTP
+limiting amounts of fluorescently labeled:
ddATP
ddGTP
ddCTP
ddTTP
What is the full name of sanger sequencing?
Sanger chain termination (dideoxy) method
Difference between dideoxynucleotide and deoxynucleotide?
dideoxynucleotide lack 3’-OH (-OH on the 3’ carbon of ribose) which prevent the next nucleotide from attaching, DNA synthesis will terminate
How can dideoxynucleotides be used to treat diseases?
azidothymidine is used to treat ends, HIV prefers AZT to normal thymidine (dTTP), DNA synthesis halts as AZT has no 3’ -OH group, side effects are mild on host cell as they prefer dTTP
What is done with dideoxylabeled ssDNA fragments in sanger sequencing?
- DNA is seperated by size in an electric field (polyacrylamide gel or capillary) using electrophoresis,
- as fluorescently labeled fragments of discrete sizes pass a detector they are activated by a laser and read by a photometer
- Read from bottom of gel to top, you will be reading the complement to your original sequence
What did the original sanger method use to label dideoxynucleotides?
radioactivity, would use 4 separate reactions for each nucleotide
What will happen if a heterozygote gene is sequenced using sanger sequencing?
there will be 50/50 color split of 2 different bps in same spot
what determines which strand is read in sanger sequencing?
the primer design