7.

013 Lecture 13: DNA Recombination I 3/4/2016

We talk about genes in three ways:
• Genetics: unit of hereditary
• Molecular Biology: DNA sequence to make product
• Recombinant DNA Technology: Gene manipulation and use

1. Recombinant DNA Technology

Use genes in “genetic engineering”
Recombinant means DNA from two or more sources, made in a lab
• many uses!
E.g. want to produce factor VIII protein (hemophilia treatment)
• 1. Isolate F8 gene
o find gene in genome
o sort out F8 gene
• 2. Make lots of F8 gene
o grow
• 3. Use to make lots of VIII protein
o transcription, translation, purify protein

2. Molecular Cloning
Cloning is making a lot of he same thing (here, DNA)
Steps to finding, cloning, expressing a gene of interest (GOI)
• Lyse cells
• Extract DNA
• Cut up DNA into GOI
• Insert DNA into “vector” (carrier with ORI)
• Sort DNA and find GOI
• Grow a lot of GOI DNA
• Transcribe, translate into protein of interest

Vectors help to carry extra DNA

• a virus that often grows in bacteria, sometimes yeast
• Carrier of extra DNA
o Has an ORI that allows it to replicate to a high copy number
§ 10,000 copies per cell
§ Gives you lots of DNA (milligrams or grams!)
ú This DNA amplifies the GOI
o Vectors can be circular or linear DNAs
 § Circular = plasmids
§ Linear = phage

When we cut DNA, we want to do it precisely, at a known place/sequence.

• This happens via restriction of endonucleases (RE)
o Bacteria have evolved this way to chop up the DNA of the invading virus
• Recognize and cut specific DNA sequences
o These can be 4, 6, 8,… bases long
o E.g. ecoRI (notice that restriction sites are often palindromic)
§ 5’GAATTC3’ à 5’G3’ + 5’AATTC3’
§ 3’CTTAAG5’ à 3’CTTAA5’ 3’G5’
• Many restricted endonucleases, each with DNA recognition site
o Blunt ends – no single stranded DNA
o Sticky ends – single stranded DNA

Pasting DNA by DNA ligase

• Creases a phosphodiester bond (as previously)
• Rules of pasting:
o Any blunt ends can paste (ligate)
§ 5’CCC3’ + 5’AAA3’ à 5’CCCAAA3’
§ 3’GGG5’ + 3’TTT5’ 3’GGGTTT5’
o Any complimentary sticky ends will ligate, meaning they must be able to
base pair
§ 5’G3’ + 5’AATTC3’ à 5’GAATC3’
§ 3’CTTAA5’ 3’G5’ 3’CTTAAG5’ (ecoRI)
• To get GOI-vector recombinant
o Cut GOI DNA (w/ RE)
o Cut vector (w/ RE)

Grow lots of GOI-vector recombinant

• Take ligation mix and put it into a bacterial host and the ORI will zip away and
you will get lots of your recombinant DNA.
• How do you get the ligation of your DNA into the bacteria?
o Use a method called transformation that either involves
§ Ca2+
§ Electric pulse
• Less than 1 DNA entering each host cell so there are methods to select the host
cells that got the recombinant DNA
o Select host cells + recombinant DNA
o Do this using the trick of having the vector having a selectable marker
§ Eg. ampicillin^R (amp^R)
§ Transform + amp à only host cells with recombinant DNA will
survive