PCR
What is PCR? (what does it stand for, what does it do?)
PCR = Polymerase Chain Reaction
-used to make many, many repeated copies of a SPECIFIC DNA sequence
-very fast
Plasmid: "small, circular molecules of DNA distinct from the bacterial chromosome"
-useful in labs for molecular cloning
What is DNA sequencing?
used to determine nucleotide sequence of an UNKNOWN stretch of DNA
True or false: Bacteria/archaea have introns, this makes it easy to sequence their genomes [gives distinct ORF]
FALSE--no introns
What is an open reading frame?
AUG-[codons]-STOP
Name the 3 steps of PCR
1) denaturation
2) annealing
3) extension
3 essential features of plasmids
1) origin or replication
2) antibiotic resistance gene (usually Amp)
3) cloning site to insert DNA of interest (MCS: "multiple cloning site")
What is the "most widely" used method of DNA sequencing?
Sanger sequencing
TRUE OR FALSE: eukaryotic genes have introns, and this makes identifying their ORFs more difficult. One strategy when sequencing eukaryotic DNA is to use mRNA sequences to locate the gene in the genome.
How would you amplify RNA [using PCR]? What would these amplified DNA copies NOT have?
You would need an enzyme that can use RNA as a template to make a DNA copy
--> this enzyme is called "Reverse Transcriptase" and the "DNA copy" that it makes is called "cDNA"
--> DNA copies would NOT have introns!
What are the 5 components of PCR?
1) template DNA
2) primers
3) dNTPs
4) Taq (heat-resistant) DNA polymerase
5) buffer
What are restriction enzymes? What types of sequences do they often cut at?
"endonuclease that cut DNA at specific sequences"
-often cut at palindromic sequences
-sticky ends vs. blunt ends
What is required for Sanger sequencing?
1) template DNA
2) primer
3) DNA polymerase
4) dNTPs
5) ddNTPs
Is gene number a good indication of biological complexity? Why/why not?
NO! Humans have more genes than flies, which makes sense, but this is NOT always the case (ex: mustard seeds > # genes than humans)
What types of things can genome sequencing tell us?
bioinformatics, computational biology/genomics, biological data mining, genome annotations
-lots of math/stats/computations required to analyze
Describe the 3 steps of PCR
1) Denaturation: DNA heated --> helix separated into 2 individual strands
2) Annealing: solution is cooled --> allows 2 primers to anneal (bind) to their complementary sequence on DNA template strands
3) Extension: DNA polymerase (taq) synthesizes new DNA strands by extending primers on each template
Describe the steps of plasmid cloning :)
1) insert DNA of interest into plasmid
2) transform plasmid into bacteria
3) grow more plasmid (what does this mean?)
What is the end result of Sanger sequencing? How do we interpret this?
synthesis of DNA chains w/ a range of sizes (why a range?), each labeled with a fluorescent dye
-Separates products produced by size
Provide explanations as to why >>> # of genes does NOT necessarily equate to more biological complexity
1) Alternative Splicing: different proteins can be made from a single gene, depending on how the mRNA transcript is spliced together-- type of post-transcriptional modification
2) post-translational modification: proteins can be combined into different complexes --> leads to many different functions
What is Illumina sequencing? What are the steps?
allows you to sequence millions of DNA fragments on a microscope slide; each spot corresponds to fragment to be sequenced
1) add ONE nucleotide
2) remove the terminating chemical group
3) read nucleotide sequence at each spot
4) use sequence overlaps to assemble whole genome sequence (lots of computing)
What are the 2 "keys" to PCR?
1) Must have sequence information of desired DNA so that you can design the primers [to be complementary to it]
2) heat-stable DNA polymerase (taq polymerase) --> allows DNA to denature without killing enzyme
What are the 2 types of repetitive sequences in eukaryotes?
1) Transposable Elements: "parasitic segments of DNA capable of moving from one location of the genome to another"
2) Short tandem repeats
-[aka microsatellites, or simple sequence repeats]: "repeating sequences of 1-5 bases"
-[aka minisatellites, variable number terminal repeats]: "repeating sequences of 6-500 bases; UNIQUE in every individual"
***minisatellites are useful in DNA fingerprinting :)
How do you sequence an ENTIRE genome?
Shotgun sequencing:
1) cut genome into smaller fragments
2) sequence each small DNA fragment
3) assemble sequence
Is there a relationship between genome size and organismal complexity?
NO!
1) polyploidy (>2 paired sets of chromosomes)
2) noncoding DNA
TRUE or FALSE: Humans and gorilla DNA sequences are 98.8% identical.
false-- CHIMPS, not gorillas :)