Molecular Biology

DNA Structure

DNA Structure

Double helix w/phosphate and deoxyribose sugar backbone on outside and nitrogenous base pairs on the inside

Monomers of DNA + base pairs and rules

Nucleotides

Adenine, Thymine, Cytosine, Guanine

A-T

C-G

Where in the cell is DNA located?

Eukaryotic: Nucleus

Prokaryotic: Cytoplasm

Function of DNA

Function of mRNA

Function of tRNA

DNA: Store genetic information/genes

mRNA: carry protein information from the DNA to the cytoplasm

tRNA: link between mRNA and chain of amino acids making up protein

RNA Structure

Single stranded (ribose sugar/phosphate and nitrogenous bases)

Monomers of RNA + base pairs and rules

Nucleotides

Adenine, Uracil, Cytosine, Guanine

A-U

C-G

Where is RNA made?

Nucleus

How do our cells make proteins from our genes?

Through transcription and then translation

Transcription - Process, Template, Enzyme, and product

Process: DNA to RNA

Template: DNA

Enzyme: RNA polymerase

Product: Complementary RNA sequence

Translation - Process, template, organelles involved, product, monomers of proteins, what do proteins do in our cells

Process: RNA to Amino Acid

Template: RNA

Organelles involved: Ribosomes

Products: Amino acids, eventually proteins

Proteins monomers: Amino Acids

Proteins Functions: “doers” of the cell

What are Codons and where are they found?

Groups of three RNA nucleotides that get read by ribosomes. One codon = one amino acid

Found in mRNA or DNA

Mutation

Change in the DNA base sequence

Point substitution mutation

One base is substituted for another

Insertion mutation

am extra base is inserted into the sequence causing a frameshift

Deletion mutation

a base is deleted from the sequence causing a frameshift

Frameshift mutation

changes the sequence of the RNA nucleotides, leading to different amino acids being produced

Effects of mutations

Silent: substitution with no affect

Missense: substitution that changes the amino acid to be different than the one produced with no mutation

Nonsense: random stop

Most significant mutation

Any frameshift because it can completely alter the entire sequence whereas sometimes substitution barely has any effects

What do we use Restriction enzymes for? How do they work? What is a restriction site

Purpose: to cut DNA, creating sticky ends

How it works:

• Enzyme finds the specific restriction site

• Cuts both ends creating sticky or blunt ends

Restriction site: where the restriction enzyme cuts the sequence

What is the difference between sticky and blunt ends? Which ones are more useful in making recombinant DNA?

Sticky ends: restriction enzyme cuts staggered, leaving one short single-stranded sequence without its complement, used to make recombinant DNA using plasmids

Blunt ends: restriction enzyme cuts directly across from each other

What do we use Recombinant DNA for? How do we make Recombinant DNA?

Purpose: genetic engineering

How it’s made:

• Cut two DNA samples with the same restriction enzyme

• Mix samples so sticky ends attract

• Ligase will join the sugar phosphate backbone of the recombinant molecule

What determines the direction and distance a substance travels on the gel by electrophoresis?

the farther away the band is from the well, the smaller the molecule; molecules with a negative charge would go to the positive electrode and vice versa

DNA Polymerase

enzyme that replicates the nucleotides with the appropriate base

Ligase

enzyme that joins two fragments of DNA together

What is a plasmid? How is it useful in biotechnology?

What? a small extra ring of DNA in the cytoplasm of bacteria

Used? to transfer foreign genetic materials into a cell

BLAST

Purpose: (search engine) To find and identify DNA, RNA, or protein sequences

How:

1. Go to website

2. Enter sequence in the search bar

3. Click on BLAST and the program will search the database

4. Analyze results

Gel electrophoresis

Purpose: To separate molecules based on size and charge

How:

1. An agarose gel is poured and sets

2. Samples are loaded into the wells

3. The gel is placed in the chamber with buffer solution

4. An electrical current is supplied and molecules move through the gel

CRISPR-Cas9

Purpose: To cut DNA at a specific and programmable sequence - we can then insert a gene of interest in that location

How:

1. Cas9 is given a guideRNA

2. Cas9 searches the DNA looking for the complementary sequence to the guide RNA

3. Once it finds the sequence, Cas9 cuts the DNA

4. Fix the gene

DNA fingerprinting

Purpose: To compare DNA samples

How:

1. Cut DNA with restriction enzymes

2. Run DNA samples on a gel  (gel electrophoresis)

3. Compare banding patterns on the gel

PCR

Purpose: To make copies of DNA

How:

1. Heat to denature

2. Cool and allow primers to anneal

3. DNA polymerase extends the new DNA nucleotide chain

4. Repeat cycle

SANGER Method (DNA sequencing)

Purpose: To determine the sequence of bases in a segment of DNA

How:

1. Replicate DNA in 4 different samples - one for each dideoxynucleotide (chain ending nucleotide)

2. Separate replicated fragments by electrophoresis

3. Read from shortest fragment to largest to know the order of the bases (from the bottom up)

Bacterial Transformation

Purpose: To use Bacteria to make proteins for medicines or other uses

How:

1. Isolate and cut plasmid with restriction enzyme

2. Cut out gene of interest with the same restriction enzyme

3. Mix DNA samples

4. DNA ligase will join the fragments together

5. Mix recombinant plasmids with Bacteria and some will take up the recombinant plasmids

6. Select for the transformed bacteria

7. They will make your protein of interest