Chapter 10: Analyzing the structure and function of genes

How are DNA clones formed? (Step 1)

A gene of interest is cleaved off by restriction enzymes that bind to a specific nucleotide sequence, that are often palindromic, resulting in DNA fragments

How are DNA clones formed? (Step 2)

The DNA fragments are isolated using gel electrophoresis and loaded into an agarose gel with an electric field applied to it, then the fragments migrate towards the positive electrode on the gel

How are DNA clones formed? (Step 3)

Once the DNA fragments are isolated, DNA ligase uses ATP to join any DNA fragments produced in vitro together to produce a recombinant DNA fragment

How are DNA clones formed? (Step 4)

Restriction enzymes will cut open a plasmid vector at a specific site and allow DNA ligase to ligate a recombinant DNA fragment to the plasmid, forming a recombinant DNA molecule

How are DNA clones formed? (Step 5)

The recombinant DNA molecule is introduced into a bacteria cell where it is then placed into a culture to grown resulting in recombinant DNA molecules being cloned multiple times as the bacteria cell multiples as well

What are most sequences recognized by restriction enzymes?

Most are symmetrical

What are the two kinds of ends that can be formed by restriction enzymes?

• Blunt ends when restriction enzymes cut in the middle of the gene of interest

• Sticky ends when restriction enzymes cut at different locations in each strand 

How does DNA fragment size gel electrophoresis?

Large fragments will migrate to the positive electrode slowly while small fragments will migrate faster, allowing for DNA fragments to be separated based on their size

How do DNA fragments with sticky ends join together?

They can join together IF their overhands have perfect base pairing with one another

what is another way sticky ends can join together?

By having enzymes come in and fill the overhands on the sticky ends, turning them into blunt ends and allows for DNA ligase to join them together

What is commonly used as cloning vectors?

Bacterial plasmids

How are genomic libraries formed?

1. A DNA molecule is cleaved using restriction enzymes, forming millions of genomic DNA fragments

2. The DNA fragments are inserted into plasmids through DNA ligase to form recombinant DNA molecules

3. The molecules are introduced into individual bacteria molecules that forms the genomic library

How is cDNA prepared from mRNA?

1. A cell culture is broken down and has its mRNA isolated

2. The mRNA is hybridized with Poly-T primer and goes through reverse transcription, forming RNA/DNA double helix and is degraded partially

3. then a ssDNA is used as a template to synthesize complementary DNA, resulting in cDNA

How does PCR allow for specific DNA fragments to be produced in a test tube?

1. A dsDNA is heated to separate its strands from each other, then specific primers bind to the strands to amplify them and are then cooled to allow for the primers to hybridize into complementary sequence on both strands

2. Afterwards, the mixture is incubated with DNA polymerase and four dNTPs to synthesize the DNA strands starting from the primers present

3. Then the process can begin again using the newly synthesized double strand products