Biology Study Material: Chapter 21 - DNA Sequencing Terminology and Techniques

DNA sequencing

Determining the exact order of the base pairs in a segment of DNA.

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HISTORY

- developed by Allan Maxam, Walter Gilbert, and Frederick Sanger (1970s)

dideoxy sequencing

AKA Chain formation

- a method of DNA sequencing that uses deoxyribonucleotides to terminate the growth of DNA strands

DNA sequencing

Determining the exact order of the base pairs in a segment of DNA.

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HISTORY

- developed by Allan Maxam, Walter Gilbert, and Frederick Sanger (1970s)

dideoxy sequencing

AKA Chain formation

- a method of DNA sequencing that uses deoxyribonucleotides to terminate the growth of DNA strands

dideoxyribonucleoside triphosphate (ddNTP)

BASIC SUMMARY

- contains a prefix di-

- 3' on lewis structure contains a H (instead of OH for normal lewis structure)

- 3' is the "chain terminator"

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MAIN IMPORTANCE:

- ddNTP's stop DNA replication

(replication stops COMPLETLY)

chain termination

the stoppage of growth of a DNA strand, RNA strand, or polypeptide sequence from a ddNTP

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RECAP: 3' contains a "H" instead of "OH" which stops elongation of DNA strand

Types of ddNTP's

adenine = ddATP

thymine = ddTTP

cytosine = ddCTP

guanine = ddGTP

Automated DNA sequencing

the use of fluorescently labeled ddNTP's and a fluorescence detector to sequence DNA

- each type of ddNTP's has a different colored fluorecent that indicates the last base in each strand

site-directed mutagenesis (SDM)

the creation of a mutant from a protein by altering a single site on the protein

- is done by mutating a single amino acid

- done in vitro (outside the cell

- done with DNA inside the plasmid

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ADVANTAGES

- allows for the alteration of a DNA sequence in a specific way

SDM restrictions

A mismatch is created:

- depending on which base is replaced, the mutant or original sequence is produced

- mutant can be identified by DNA sequencing

Why is SDM performed?

allows researchers to see how mutation affects

- the expression of the gene

- the function of a protein

- the phenotype of an organism

Blotting techniques

- Northern blotting (RNA)

- Southern blotting (DNA)

- Western blotting (Protein)

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KEY FEATURES:

- all are quantitative

- uses electrophoresis

What are blotting techniques used for?

- is used to detect mRNA and proteins

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HOW IS IT VISUALIZED?

- ETBR

EtBr (ethidium bromide)

planer molecule that intercalates with DNA and makes them visible in orange after being exposed to UV light.

Northern blotting (RNA)

- used to identify specific RNA within a mixture of many RNA molecules

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FEATURES:

- uses RNA for electrophoresis

- uses agarose gel the contains formaldehyde

- uses radioactive sscDNA probes

Formaldehyde

the chemical used to break down hydrogen bonds in RNA strands

What can Northern Blotting determine?

- if a specific gene is transcribed in a particular cell type or is in a specific stage of development

- it can reveal if a pre-mRNA is spliced

how is Northern Blotting similar/different to RT-qPCR?

Similar:

- both are used to quantify the amount of RNA is spresent for a specific gene of mRNA

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Difference:

- RT-qPCR works for both RNA and DNA

- Northern blotting uses its length instead of its amount to transcribe a gene

Northern Blotting Procedure

1. RNA is extracted from the cells and is then purified

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2. its then separated by gel electro.

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3. its bottled onto nitrocellulose or nylon filters

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4. Filters are placed into a solution containing a radioactive probe

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5. Filters are then exposed to x-ray film

where they can be detected as complementary radiolabeled probe/ dark bands on

spliced

process of removing intron in the nucleus of the cell

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WHAT DOES IT MEAN ON AN AGE MACHINE?

- the bands are separated on one lane

Southern Blotting (DNA)

separating DNA molecules based on size

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FEATURES:

- electrophoresis uses dsDNA

- uses agarose gel (by seeing multiple smears)

- uses a radioactive sscDNA probes

smears

indicates that there is a variety of possibilities of different fragment sizes

What does Southern Blotting determine?

- main use is to identify a specific gene or fragment of DNA

Southern Blotting Procedure?

BASED ON NOTES:

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1. Extract and purify DNA from cells

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2. cut DNA into different sized fragments using restriction endonucleases

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3. Perform gel electrophoresis (to allow the DNA fragments to separate)

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4. Denature the DNA

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5. transfer to nitrocellulose paper (where blotting will occur)

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6. add labeled probe for hybridization to take place

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7. wash off unbound/excess probe

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8. autoradiograph the sample

Restriction endonucleases (enzymes)

RECAP:

molecular scissors that can cut DNA in specific locations

Nitrocellulose

a modified cellulose molecule used to make paper membrane for blots of nucleic acids and proteins

Autoradiography

a procedure that locates radioactive substances in a slice of tissue

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the radiation exposes a photographic emulsion or a piece of film that covers the tissue

Western blotting (Proteins)

- used to identify a specific protein within a mixture of many protein molecules

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FEATURES:

- electrophoresis uses protein

- gel is polyacrylamide

- uses either folded or denatured proteins

- uses antibodies as its probe

polyacrylamide

A polymer used as a gel material in vertical electrophoresis

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is used to used to separate small proteins apart

what can western blotting determine?

it can determine if:

- a specific protein is made in a particular cell type or is in a particular stage of development (similar to Northern blotting)

Western blotting procedure

BASED ON NOTES:

1. dissolve detergent sodium dodecyl sulfate

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2. separate the "-" charge proteins by polyacrylamide gel electrophoresis

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3. place sample onto nitrocellulose or nylon filters

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4. place the filter in a solution containing the primary antibody

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5. then place filter in a solution containing a secondary antibody

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6. add colorless XP to the sample to allow an appearance of dark bands on electrophoresis

sodium dodecyl sulfate (SDS)

detergent that denatures proteins and coats them with a negative charge

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prefix dode- = 12

Polyarcylamide gel electrophoresis

method of separating proteins depending on:

- size

- structure

- molecular weight

primary antibody

Antibody that recognizes protein of interest

secondary antibody

antibody that is conjugated to alkaline phosphatase

- recognizes the constant region of the primary antibody

methods for analyzing DNA and RNA binding proteins

- electrophoretic mobility shift assay

- DNAse I footprinting

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PURPOSE?

- both methods find what protein is involved in transcription factors

Electrophoretic Mobility Shift Assay (EMSA)

AKA: gel shift assay / gel retardation assay

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WHAT DOES IT DETERMINE?

- if a protein binds to a specific DNA fragment or RNA molecule

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FEATURES:

- is ran under native gels

- uses a radioactive dsDNA probe

- commonly looks at 20 base pairs per run

native gels

Polyacrylamide gels:

- acrylamide gels with no SDS

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Agarose gels:

- agarose gels with no denaturing techniques

EMSA restrictions (No denaturing techniques)

must be performed under non-denaturing conditions

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- buffer and gel should not cause the unfolding of the proteins nor the separation of the DNA double helix

how does EMSA work

1. DNA and protein molecules migrate through a gel matrix at different rates

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2. Small DNA oligomers will run quickly through a gel

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3. proteins migrate more slowly

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4. DNA protein complexes are larger than either - we see a shift in migration

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5. monitor the migration of free DNA to protein-bound DNA

DNase I footprinting assay

AKA: DNA footprinting / DNase footprinting

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WHAT DOES IT DETERMINE?

- a harder assay that shows detailed interactions between a protein and DNA

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FEATURES:

-DNA fragment used is typically around 300 base pairs (BP)

How does DNase I footprinting work?

1. probe with 300 BP is incubated with NL solution

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2. add DNase I to make a single cut on each probe

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3. rub it on a polyacrylamide gel (to denature urea)

CRISPR-Cas technology

WHAT DOES IT DO?

- allows you to make/insert genes in a living cell

- targets restriction enzymes to create fragments

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ADVANTAGES:

- uses a noncoding RNA from a microbe that doesn't have a specific RNA sequence

types of non coding RNA's

- tracrRNA

- crRNA

tracrRNA

RNA needed to associate with crRNA and Cas9 for function of the enzyme (crRNA binds to Cas9)

Cas9

RNA-guided DNA endonuclease enzyme associated with the CRISPR

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MAIN FUNCTION:

- produce a ssDNA

- can cut target DNA

crRNA

RNA transcribed from CRISPR tech. that binds to a target DNA

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MAIN FUNCTION:

- its a protein complex that degrades complementary invading viral nucleic acid

sgRNA (single guide RNA)

a synthetically engineered binding site that links/binds Cas9 to a gene of interest

cas9 repair events

- nonhomologous end joining (NHEJ)

- Homologous recombination repair (HRR)

Nonhomologous end joining (NHEJ)

the region that may incur a small deletion that inactivates the gene

- loses information

- cripples/breaks a gene

homologous recombination repair (HRR)

a repairing of double-strands that occurs when the DNA strands from a sister chromatid are used to repair a lesion in the other sister chromatid