molecular bio lecture 9

technical breakthroughs made in biology

rapidly determine the nucleotide sequence of entire genomes, determine the function of genes and proteins in living organisms, manipulate DNA and influence understanding & treatment of disease and mutations, and production of pharmaceuticals

how can a snapshot of gene expression be obtained?

through analysis of mRNA

illumina sequencing

sequences DNA fragments in clusters; allows for rapid sequencing

illumina sequencing part 1

DNA is fragmented

Fragments are then attached to solid support

Fragments are amplified using PCR to make multiple copies

Results in DNA clusters

illumina sequencing part 2

1,000 identical DNA fragments are anchored to solid support

illumina sequencing part 3

DNA polymerase adds nucleotides to the growing DNA strand

Each nucleotide is attached with a fluorescent tag and chemical group that blocks further elongation

illumina sequencing part 4

photo is taken of slide after each nucleotide is added

fluorescent tag that corresponds to one of four bases allows the identity of the added nucleotide to be recorded

illumina sequencing part 5

after each nucleotide is added/recorded, fluorescent tag and chain-terminating chemical group are removed

DNA polymerase continues adding next nucleotide

illumina sequencing part 6

information obtained is used to reconstruct the sequence of the original DNA fragment

how to understand which genes are being expressed?

by analyzing the mRNAs that are being produced by the cell

RNA seq p1

obtain RNA from cell of interest

RNA seq p2

fragment RNA which makes it easier to sequence

RNA seq p3

convert fragmented RNA into cDNA using reverse transcriptase which creates stable DNA copy which can be sequenced

RNA seq p4

process cNDA to prepare a sequencing library

RNA seq p5

amplify cDNA library using PCR to make enough copies of each cDNA fragment for sequencing

RNA seq p6

cDNA library is loaded into sequencing machine (like illumina) where cDNA can be sequenced

purpose of single cell RNA sequencing

to catalog the mRNAs produced by individual cells

how are the mRNA from each cell processed in single cell RNA sequencing

mRNA from each cell is converted into cDNA and each cDNA it tagged with its own barcode

What can single-cell RNA-Seq help identify?

It can identify genes that are coordinately regulated

How can single-cell RNA-Seq be useful in analyzing gene expression?

It can be used to analyze changes in gene expression in individual cells under different conditions

what does barcode in single cell RNA sequence do?

allows for identification of mRNA from each individual cell in the sequencing process

in-situ hybridization of mRNA purpose

allows for specific nucleic acid sequence to be visualized in its normal location within a cell or tissue

purpose of using labeled probes in in-situ hybridization

used to detect complementary nucleic acid sequences in a cell, tissue, or organism

fluorescence in situ hybridization

method of in situ hybridization that uses fluorescent probes to detect specific DNA or RNA sequences

what does ribosome profiling determine

which mRNAs are being actively translated to produce proteins

what is done to cell or tissue extract in ribosome profiling

the extract is exposed to ribonuclease which digests unprotected RNA segments

what happens to the RNA segments protected by ribosomes during ribosome profiling

protected RNA segments are released from the ribosomes, converted into complementary DNA, and then sequenced

one of the best ways to determine the function of a gene?

what happens to an organism when the gene is inactivated

classical approach to studying gene function in the past?

the selection of random mutants to find unusual phenotypes, known as classical genetics

more recent approach to studying gene function?

targeted selective inactivation of genes of interest, called reverse genetics

what’s RNA interference (RNAi)?

the introduction of a double-stranded RNA (dsRNA) molecule into a cell or organism that matches the gene sequence to be inactivated.

What happens to the dsRNA after it is introduced into a cell during RNAi?

The dsRNA is cleaved and processed by RNAi machinery to produce shorter double-stranded fragments called small interfering RNAs (siRNAs)

What do siRNAs do in RNA interference?

___, which are single-stranded RNA fragments, hybridize with target gene mRNAs and direct their degradation

What does targeted gene replacement involve in vitro

___ involves changing or altering the regulatory region of a gene or introducing changes to its coding sequence.

What happens after the gene is altered in vitro during targeted gene replacement?

The altered gene is reintroduced back into the organism to determine its function.

transgenic organism

____ is one that has been modified by the introduction of a foreign gene, called a transgene.

What is a mutant organism in the context of targeted gene replacement?

A ___ is one in which the gene has been altered and expresses a new phenotype, created by the targeted gene replacement.

What is the purpose of creating conditional knockout mice?

___ allow for the disruption of a known gene more selectively in a particular cell type or at a certain stage of development.

Why are conditional knockouts useful for studying genes critical during embryonic development?

___ are useful because they allow researchers to study genes that are critical during embryonic development without disrupting the gene throughout the entire organism.

How does a conditional knockout mouse work?

it requires an enzyme that can be directed to excise and inactivate the gene of interest at a specific time or in a specific tissue.

• Q: What is the main purpose of DNA cloning?

allows the production of recombinant protein in large quantities

How is efficient protein production accomplished in DNA cloning?

Efficient production is usually accomplished using specially designed expression vectors.

Q: What do expression vectors contain to ensure high-level gene expression?

Expression vectors contain transcription and translation signals that direct the inserted gene to be expressed at high levels.

What are vectors/plasmids?

Vectors/plasmids are circular recombinant double-stranded DNA (dsDNA) molecules.

Q: What are modified bacterial plasmids used for in DNA cloning?

Modified bacterial plasmids are used as cloning vectors and expression vectors in DNA cloning.

cloning vector

DNA molecules that you can insert foreign DNA into

• Q: What is included in each plasmid for efficient cloning?

• A: Each plasmid contains a replication origin, which allows for the production of large amounts of the plasmid within bacterial cells, and a selection marker.

• Q: What is the role of the selection marker in plasmids?

• A: The selection marker allows researchers to identify and select cells that have successfully taken up the plasmid.

• Q: What do restriction enzymes/restriction nucleases do?

• A: They cleave double-stranded DNA (dsDNA) at specific nucleotide sequences.

• Q: What are the target sequences for restriction enzymes?

• A: The target sequences are typically short and specific, often palindromes.

• Q: What are "sticky ends" in the context of restriction enzymes?

• A: "Sticky ends" are single-stranded overhangs generated when a restriction enzyme cuts DNA at specific sequences.

• Q: How does the restriction enzyme HaeIII function?

• A: HaeIII cuts at a sequence of four nucleotide pairs.

• Q: How often would a restriction enzyme with a target sequence of four nucleotides cleave by chance?

• A: It would cleave every 256 nucleotide pairs (1 in 44) purely by chance.

• Q: What was discovered to aid in isolating and cloning DNA molecules?

• A: A class of bacterial enzymes that cut double-stranded DNA (dsDNA) at a particular sequence was discovered.

• Q: How are specific DNA fragments produced from any genome?

• A: The bacterial enzymes cut dsDNA, producing a reproducible set of specific DNA fragments.

• Q: What is the process used to amplify a desired DNA fragment?

• A: The desired fragment is amplified by DNA amplification, producing many identical copies.

• Q: Why is DNA amplification important in cloning?

• A: DNA amplification makes it possible to separate a gene of interest from the rest of the genome.