Theme 4 - Module 3 Flashcards
How has PCR revolutionized the world of cellular and molecular biology?
Shed light on:
- diagnosis of genetic defects
- detection of viral DNA in cells
- producing large amounts of DNA from fossils containing trace amounts of DNA
- able to link specific individuals to DNA samples during forensic investigations
Taq polymerase was first isolated from which bacterial species?
Thermus aquaticus
adapted to live in hot springs
True or false: gel electrophoresis is a technique that can only be used with separated DNA fragments from PCR
False
Other sources (other than PCR) can also supply separated DNA fragments
True or false: gel electrophoresis can only separate DNA fragments, not any other molecule
False
Can separate other macromolecules including RNA and proteins
Why are DNA and RNA are negatively charged molecules?
Bc of their ionized phosphate groups along the phosphodiester backbone
True or false: the bands would NOT be visible while separating in the gel electrophoresis apparatus
True
True or false: gel electrophoresis can only be used on single size of DNA molecule
False
Can be used to separate and visualize a DNA sample containing a mixture of DNA fragments of different sizes
What was a limitation to Sanger’s DNA sequencing technique?
It could only determine the sequence of small fragments of DNA
What are the three phases of shotgun sequencing?
(after breaking the entire genome into different sized pieces)
Phase 1: Random sequencing of the DNA in each fragment
Phase 2: Identifying the regions of overlap between the generated fragments and assembling the sequence of nucleotides that makes up each chromosome
Phase 3: Annotating the sequences and identifying the coding regions, regulatory regions and non-coding regions
What was the first bacterial whole genome to be sequenced?
Haemophilus influenza
What was the first multicellular organism genome to be sequenced? The second?
1) C. elegans
2) Drosophila
What was critical to the process of whole-genome sequencing?
The development of computational software capable of facilitating the assembly of the fragments
What is the purpose of putting dideoxynucleotides within the tubes as well? (shotgun seq)
ddNTPs are missing the -OH group at the 3’ position. Therefore, would not allow for further elongation of a growing DNA strand and would terminate replication. Leads to a series of interrupted daughter strands.
True or false: insertion of ddNTPs or the dNTPs is a organized and calculated process
False
It’s random process. Many fragments of many different sizes can potentially terminate at every possible nucleotide
True or false: there is a smaller amount of ddNTPs that are added relative to the amount of dNTPs
True
How do you distinguish all the chain terminators that are present in all the replicated DNA fragments?
By labelling each of the four ddNTPs with diff coloured fluorescent dyes
What is a limitation to the Sanger dideoxy chain-termination method?
Can only determine the sequence of fragments of DNA up to several hundred nucleotides in length
What are contigs?
Overlapping DNA segments that are assembled into a consensus region of DNA
For any double-stranded DNA sequence, how many possible reading frames are there?
6
What characteristics of a seq indicates that its a good reading frame?
Long, continuous triplets of nucleotides that specify the amino acids that will make up the protein. Lacks a stop codon and are flanked on either side by a start and stop codon
(also indicates that that may be the coding sequence)
True or false: computer programs only look for coding regions
False
Also look for typical sequences that code for promoters, or other regulatory sites
True or false: it’s hard to scan and identify regions of interest on both prokaryotic and euk seq DNA
False
Prokaryotic genomes can be easily scanned
Eukaryotic genomes need an array of different techniques to find the introns and exons especially
How would you know if a particular sequence of DNA coded for tRNA?
tRNA molecules forms hairpin structures in which the molecule folds back on itself and undergoes complementary base pairing within itself
Therefore, look for nearby complementary sequences within the sequence
True or false: 50% of the average eukaryotic genome consists of repeated sequences that do not code for functional gene products
True
