Lecture 03 | 09/13 Genomic Approaches for Analyzing Nucleic Acids

2022/09/13 10:27 AM posted in  BIO105   comments
Tags:  #BIO-105

A Cloning Problem

Part A: Using this information, what must have happened during the cloning process?

In the normal situation, there will be a sticky end and a blunt end, so the vector will not be self ligate. But in this one, two ends are both sticky.
The LacZ gene will be self ligated and reform a vector. So it will still be blue.

Part B: If you isolate the plasmids from the blue bacterial colonies and sequence them, what do you predict will be the base sequence of the top DNA strand at the ligation site?


PCR and DNA Sequencing

The main priciple of PCR

Combinig Cloning with PCR

Use restriction enzyme to cut target piece off the strands

Clone from mRNA

Use reverse transcriptase to make cDNA from mRNA.

Real-time PCR(Quantitative PCR, Q-PCR)

  • Measures amount of product after each cycle.
  • Product produces a fluorescen signal

    CT: cycle threshold.
    We can use it to quantify how much in our original sample.

Poll Everywhere

D.8 Because in every cycle, the amount of genes will double.

Different Q-PCR Techonologies


SYBR-green works really well when we just have a very specific single product.
Inexpensive but binds to all dsDNA. Only works if product is specific.


Expensive, but very specifc.

First Generation(Sanger) DNA Sequencing: relies on dideoxynucleotide terminators

Conceptually similar to endpoint PCR.

  • Requires four reactions, a large gel and radioactivity.
  • Gives only 100-200 base pairs per sequencing run.

DNA Sequencing Problem

7 As and plus the whole one.
dNTPs: dATP, dCTP, dGTP, dTTP.

3 = 2 + 1