DNA sequencing, hybridization techniques and transcription (Dr. Ruddy)

Question 1

What is the Sanger method ?

Answer 1

Sanger sequencing is a method developed by Frederick Sanger in 1977 based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs, where N can by any nt) by DNA polymerase during in vitro DNA replication. With the 3’-OH no longer present, it can longer initiate a nucleophilic attack on the alpha phosphate of another nucleotide and thus stars stops growing.

Question 2

How can the Sanger method be used to sequences DNA fragments ?

Answer 2

The ddNTPs may be radioactively or fluorescently labeled for detection in automated sequencing machines. The ddNTP is added to be approximately 100-fold lower in concentration than the corresponding deoxynucleotide, allowing for enough fragments to be produced while still transcribing the complete sequence. Of course, 4 seperate reactions need to be carried out for the 4 nts. The fragments is then digested by restriction enzymes and analyzed by gel electrophoresis and autoradiography.

Question 3

What is fluorescence based sequencing (or the dye terminator method) ?

Answer 3

In fluorescent DNA sequencing, dyes are attached to the dDNTPs. The DNA fragment in the sequencing reaction becomes dye-labeled when a ddNTP is incorporated. The dye terminator method uses a single reaction tube (rather than four) because each dideoxynucleotide is associated with a different dye. The cost and time for sequencing are therefore reduced, making this approach the preferred method used by most laboratories.

Question 4

What is shotgun sequencing ? (bonus)

Answer 4

Shotgun sequencing is a sequencing method designed for analysis of DNA sequences longer than 1000 base pairs, up to and including entire chromosomes. This method requires the target DNA to be broken into random fragments. After sequencing individual fragments, the sequences can be reassembled on the basis of their overlapping regions. An excitation laser shines through the capillary and the light emitted by the fluorescent dye as it returns to a lower energy level is detected by a detector system. As each coloured band is detected, it creates a signal which is processed by the sequencer and presented as a peak on a graph. Each peak represents a different base.

Question 5

How efficient is next generation sequencing compared to the Sanger method ?

Answer 5

Next generation sequencing can read 1Gb of DNA (1/3 of Genome) in half a day. This took the Sanger method over a year.

Question 6

What are the three main types of RNA ?

Answer 6

mRNA, tRNA and rRNA.

Question 7

What is the role of mRNA ?

Answer 7

It encodes the protein to be translated.

Question 8

What is the role of tRNA ?

Answer 8

tRNA is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length, that serves as the physical link between the mRNA and the amino acid sequence of proteins.

Question 9

What is the role of rRNA ?

Answer 9

rRNA fabricates the polypeptides and provides a mechanism for decoding mRNA into amino acids and interacts with the tRNA during translation.

Question 10

Where are RNA’s made ?

Answer 10

In the nucleolus of the cell (in the case of eukaryotes). Special enzymes synthesis the different forms of RNA.

Question 11

What does RNA polymerase I transcribe ?

Answer 11

Pre-rRNAs (ribosome components, proteins synthesis).

Question 12

What does RNA polymerase II transcribe ?

Answer 12

mRNAs (encodes proteins), snRNAs (RNA splicing), miRNAs (post-tranlational gene control)

Question 13

What does RNA polymerase III transcribe ?

Answer 13

tRNA (protein synthesis), 5S RNA (ribosome component, protein synthesis), snRNA U6 (RNA splicing), 7S RNA (signal-recognition particle for insertion of polypeptides into the endoplasmic reticulum), other stable short RNAs (various functions, unknown for many).

Question 14

What is a promoter ?

Answer 14

A promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA.

Question 15

What is the TATA box ?

Answer 15

The TATA box (also called the Goldberg-Hogness box) is a DNA sequence (TATAAAA) found in the promoter region of genes (aprox 25 bp upstream the start site) in archaea and eukaryotes; approximately 24% of human genes contain a TATA box within the core promoter. Transcription factor TFIID binds to the TATA box.

Question 16

What is the CAAT box ?

Answer 16

The CAAT box is a distinct pattern of nucleotides with GGCCAATCT consensus sequence that occur upstream by 60-100 bases to the initial transcription site. The CAAT box signals the binding site for the RNA transcription factor CTF, and is typically accompanied by a conserved consensus sequence.

Question 17

What is the GC box ?

Answer 17

Th GC box is a distinct pattern of nt (GGGCGG) that acts in a similar fashion to an enhancer sequence, binding proteins called Sp1s which further stabilise the preinitiation complex at the promoter of a gene, and thereby increase the rate of transcription of that gene. GC boxes are generally located about 110 bp upstream of the transcription initiation site.

Question 18

What is an enhancer ?

Answer 18

A enhancer is a short (50-1500 bp) region of DNA that can be bound by proteins to increase the likelihood that transcription of a particular gene will occur. These proteins are usually referred to as activators or transcription factors.

Question 19

What is an enhanceosome ?

Answer 19

The enhanceosome is a higher-order protein complex assembled at the enhancer and regulating the expression of a target gene. The binding and assembly of the activating proteins is cooperative due to energetically favorable protein-protein interactions formed in this complex.

Question 20

What is a coactivator ?

Answer 20

A coactivator is a protein that increases gene expression by binding to an activator (transcription factor) which contains a DNA binding domain.

Question 21

How can the DNA bring the activators closer to the core promoter ?

Answer 21

By bending.

Question 22

What are the steps for the initiation of transcription by RNA polymerase II ?

Answer 22

Activator proteins (transcription factors) bind to do DNA control elements within an enhancer. The DNA bends to bring the activators close to the core promoter regions containing the TATA box. The activators now form an enhanceosome. Coactivators link the enhanceosome to TFIID (a TATA binding protein), helping to positing TFIID on the TATA box. Other general transcription factor and RNA polymerase II binds. Transcription can begin.

Question 23

How many domains do transcription factor at least contain ?

Answer 23

2: a DNA binding domain and one or more activation (or repression) domains.

Question 24

What transcription factor regulates the levels of cortisol in the blood ?

Answer 24

The glucocorticoïd receptor.

Question 25

What are typical DNA-Binding Domains motifs ?

Answer 25

The helix-turn-helix motif (the lambda repressor, the trp and lac repressor), the zinc finger motif, the leucine zipper motif, the helix-loop-helix motif.

Question 26

What is CRP (C-reactive Protein) ?

Answer 26

C-reactive protein (CRP) is an annular (ring-shaped), pentameric protein found in blood plasma, whose levels rise in response to inflammation. Normal range = 1-3mg/L.

Question 27

What are the three key processes before producing a final mRNA strand in eukaryotes ?

Answer 27

Capping, slicing and polyadenylation. A 7-methylguanosine cap is added to the 5’ end of the pre-mRNA while elongation is still in progress. The 5’ cap protects the nascent mRNA from degradation and assists in ribosome binding during translation. After splicing, introns are removed and exons are ligated. A poly(A) tail (a stretch or multiple AMPs) is added to that RNA at the end of transcription. The poly(A) tail protects the mRNA molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and translation.

Question 28

Are most eukaryotic genes moncystronic of polycistronic ?

Answer 28

Mostly monocystronic. A eukaryotic gene contains the genetic information to translate only a single polypeptide. On the other hand, most of the mRNA found in bacteria and archaea is polycistronic, as is the human mitochondrial genome.

Question 29

What are Cis-regulatory elements (CREs) ?

Answer 29

Cis-regulatory elements (CREs) are regions of non-coding DNA which regulate the transcription of nearby genes.

Question 30

What are Trans-regulatory elements (TRE)s ?

Answer 30

Trans-regulatory elements are genes which may modify or regulate the expression of distant genes. More specifically, trans-regulatory elements are DNA sequences that encode transcription factors.

Question 31

What is an operon ?

Answer 31

An operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter. This is mostly the case in prokaryotes (because most eukaryotic genes are monocistronic) but is also seen in C. Elegans and Drosophila melonogaster.

Question 32

What can reverse transcriptase do ?

Answer 32

Translate the mRNA into cDNA (complementary or copy DNA).

Question 33

What is a microarray and what is it used for ?

Answer 33

A DNA microarray (DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. DNA microarrays can be used to detect DNA, or detect RNA (most commonly as cDNA after reverse transcription) that may or may not be translated into proteins. The process of measuring gene expression via cDNA is called expression analysis or expression profiling.

Question 34

As many as 60 proteins may congregate at a transcription initiation site (activators, repressors, etc.). What does such complexity allow ?

Answer 34

A fine and sophisticated tuning of gene expression. In eukaryotes, about 7% of all genes code for DNA binding proteins.