Recombinant DNA and PCR

(CRISPR) Clustered regularly interspaced short palindromic repeats

(CRISPR) Clustered regularly interspaced short palindromic repeats

Jennifer Doudna and Emmanuelle Charpentier

• Breakthrough of the yr 2015

CRISPR RNA (crRNA)

Evolved to cut up foreign viral and plasmid DNA

CRISPR-Cas9

Engineered system is derived from Streptococcus pyogenes bacteria

crRNA and other RNA molecules engineered into a single guide sgRNA

8-12 bp seed sequence custom made to pair with any DNA sequence

Effector complex

• Recognizes PAM sequence

• Unwinds DNA

  • Allowing seed sequence to bind to target- Cas9 then cuts DNA

DNA repair mechanisms

Used to disable gene

Insert donor DNA via homologous recombination

PROS of CRIPSR-Cas9

sgRNA can be modified to edit almost any place in the targeted genome

Used in intact cells

CONS of CRIPR-Cas9

Sometimes difficult to import system into live cells- Cas protein is large

Off-target cleavage from mismatched sgRNA

Does not have 100% efficiency in a multicellular organism- creates genetic mosaics (some altered, some other cells not)

Cloning: Biology vs genetics

Biology: Process of producing populations of genetically identical individuals

Genetics: Creating copies of DNA fragments, and sometimes, the protein products of the DNA

Essential to study genomes

Basic Cloning Tool

DNA from organism

Restriction enzyme (endonucleases)

• Cut DNA at specific recognition sites

DNA ligase

• Joins recombinant DNA molecules permanently

Cloning vectors

• Vehicle for replication of foreign DNA

Host microorganism (often bacteria)

• Factory for propagation of recombinant DNA in vector

Restriction enzymes 1

• Naturally in bacteria

• Cut out viral DNA that infects genome, thus restricting access of the virus

• Recognize specific DNA sequences in the genome called restriction sites, often palindromes

• Cleave phosphodiester bonds, leaving a 5’ phosphate and 3’ hydroxyl group in the extracted fragments

Abt 400 known

Named by 1st letter= genus

2 and 3rd= species

Additional letters are for strains

Palindromes

Mirror images of the same sequence

Methylated DNA

Protects bacterial genome from RE’s

RE Probability

4 base pair possibilities

(1/4)^n, where n=number of bases in restriction site

Small restriction site = many pieces of DNA of short length

RE Cutting: Blunt ends

RE cuts both strands of DNA between same two base pairs

RE Cutting: Sticky (overhanging) ends

RE’s make staggered cuts in DNA

Sticky ends significance

Fragments of DNA cut by same RE with same sticky ends both with each other easily.

Ligase cements these bonds

New DNA molecule is recombinant

Recombinant DNA has the gene of interest

Plasmid vectors

Circular DNA and replicate separately from bacterial chromosomes

Engineered to have:

• Ori sequence: needed for independent replication in bacteria

• Selectable marker: to identify bacteria with the plasmid (antibiotic resistance for example)

• At least one unique restriction site for insertion of recombinant DNA

pUC19- A great vector

1. 100 copies per cell

2. amp^R selectable marker (ampicillin resistance)

3. Multiple cloning site (MCS) with several unique restriction sites

4. MCS located in a coding gene called B-galactosidase (lacZ+)

   • Gene functions normally if there is no DNA cloned into the MCS, but any cloned DNA will destroy gene function

   • A chemical called X-gal indicates if bacteria have normal gene function (blue) or inactive gene (white)

PCR History

Invention: Kary Mullis (1944-2019)

Founded a company to sell jewelry with DNA of famous dead people like Elvis and Marilyn Monroe

Extraterrestrial visitors: green raccoon 1985

PCR Functions

Make many copies of DNA that is supplied as a template

Amplified fragments can be sequenced, cloned, probed, or sized using electrophoresis

PCR Importance

1. Cloning for DNA sequencing

2. DNA-based phylogeny (relationships)

3. Diagnosis of genetic/infectious disease

4. Genetic fingerprinting

PCR How it works

Unwinding of DNA (melting)

Priming

Polymerization

Components of a PCR Reaction

Buffer (Mg)

Template DNA

2 Primers that flank the fragment of DNA to be amplified

dNTPs

Taq DNA polymerase

Taq DNA Polymerase

Archaea that lives in hot springs at 70 C

Yellowstone national park

Drawbacks of PCR

Taq polymerase has no proofreading ability, BUT some dif. kinds of DNA polymerase for PCR can minimize error rates

If mismatch error occurs EARLY in the PCR process, mistake can be magnified millions of times, giving a mistake in the sequence.

Gel electrophoresis

Phosphate groups on DNA molecules have a negative charge, so they are pulled toward positive end of gel

PCR Temperatures and repeats

• Melting: 94 C

• Annealing primers: 50 C

• Extension: 72 C

• Melting: 94 C

Repeat 30x