Methods Course - DNA Cloning

What is the goal/target for DNA cloning?

to insert a specific piece of DNA (a gene or other DNA sequence) into a plasmid (small circular piece of DNA found in bacteria) to study or use it in various experiments.

What is the purpose of genomic/sequencing constructs?

add new DNA to understand genes

these constructs are used to add new DNA sequences to the plasmas, this can help in sequencing and understanding the genome or specific genes.

What is the purpose of expression constructs?

make proteins from inserted DNA

designed to help cell make specific protein, DNA sequence inserted into plasmid is transcribed into mRNA and then translated into a protein.

What is the purpose of reporter constructs?

use reporter genes to see gene activity

contain a “reporter gene” (like GFP which makes cells glow green) to study where and when a gene is active. they help scientists see how genes are expressed in different tissues or conditions.

Simplified Example of DNA cloning:

1. choose a vector (plasmid) - a circular piece of DNA that can replicate in bacteria

2. insert DNA of interest (insert) - add the gene you want to study into the plasmid

3. transform bacteria - introduce the plasmid into bacteria so they can replicate it

4. analyze - study how the inserted DNA behaves (protein production/gene expression/)

How to generate a useful DNA fragment - “Insert”

1. restriction digest: method to cut DNA at specific sequences using enzymes called restriction enzymes

   1. types of cuts:

      1. sticky ends: cuts made by enzymes like EcoRI that leave overhanging ends (go well with complementary sequences)

      2. blunt ends: cuts made by enzymes like EcoRV that leave no overhangs

   2. partial/complete/double digest

      1. partial - only some of the restriction sites are cut

      2. complete - all extraction sites are cut

      3. double digest - uses two different restriction enzymes to cut at two different sequences

2. cloning process:

   1. donor plasmid - contains the DNA fragment (insert) you want to clone

   2. restriction digest - use enzyme to cut the donor plasmid and the recipient plasmid

   3. insert DNA - DNA fragment (insert) from the donor plasmid is inserted into the recipient plasmid

   4. ligation - the DNA fragment is joined into the recipient plasmid using an enzyme called DNA ligase

3. PCR:

   1. technique used to amplify specific DNA sequences

   2. adding adaptors/handles - small sequences of DNA (adaptors) are added to the ends of the DNA fragment to make it easier to clone

4. Shearing:

   1. method to randomly break DNA into smaller pieces to create genomic libraries to avoid biases that come frm

1. restriction digests

2. stick vs blunt ends

3. PCR

4. shearing

Example:

1. cut the DNA

2. insert the DNA

3. seal the DNA

4. amplify the DNA

Type of restriction digest:

Sticky ends w/EcoRI

Type of restriction digest:

blunt ends: EcoRV

What are the minimal parts required for plasmid?

1. origin of replication (ori) - ensures plasmid replication

2. selectable marker - IDs cells with the plasmid (antibiotics/ampicillin or nutritional/URA)

3. multiple cloning site (MCS) - provides sites for DNA insertion

Cloning Plasmid

1. purpose: clone or amplify a specific DNA fragment

2. key features: MCS, selectable marker, ORI

   1. MCS - essential with several restriction sites for easy insertion of DNA fragments

3. When to use:

   1. amplifying or cloning specific DNA fragments

   2. preparing DNA for sequencing or other analyses

4. Example: cloning a gene to study its sequence or to use it in further experiments

Expression Plasmid

1. Purpose - produce a specific protein in a host cell

2. key features:

   1. strong promoter - to drive high level of gene expression

   2. RBS - ensure efficient translation of mRNA into protein

   3. selectable marker - ID cells that have taken up plasmid

   4. MCS - included for inserting the gene of interest

   5. transcriptional terminator - ensures proper termination of transcription

   6. tags/fusions (optional) - (His-tag for easy purification or detection)

3. When to use:

   1. protein production for study, industrial applications or therapeutic purposes

   2. functional studies to understand protein roles

   3. protein purification

4. Example: producing insulin in bacteria for diabetes treatment

Reporter Plasmid

1. Purpose: study promoter activity and gene regulation

2. key features:

   1. promoter region - main feature, can be weak or strong depending on study

   2. reporter gene (GFP) - often included

   3. selectable marker - ID cells that have taken up plasmid

   4. MCS - included for inserting different promoter or regulatory sequences

3. when to use:

   1. analyzing promoter strength and activity

   2. investigating gene regulation

   3. studying tissue-specific expression or response to eviormental changes

4. Example: studying how a certain promoter responds to different temperatures using a reporter gene like GFP.

Transformation

introducing DNA into bacterial cells using chemicals or electric shock

transfection

introducing DNA into vertebrate cells using calcium phosphate, lipsomomes, or electric shock

selection

killing off or separating cells to find ones with the new DNA

screening

using visible changes or sequential testing to ID cells with new DNA