Exam 2 Review Flashcards
What are plasmids?
Small circular DNA
What can plasmids be used as?
Cloning vectors
What are restriction endonucleases?
Enzymes that cut dsDNA at specific recognition sites
What type of sequence do almost all restriction enzymes cut?
Palindrome sites
What is meant by palindrome?
Palindromes are sequences read the same forwards on the sense strand as it reads backwards on the antisense strand
Example:
5’ -CGATCG- 3’
3’ -GCTAGC- 5’
(5’ -> 3’ = CGATCG)
What is transformation?
Heat shocking bacterial shells to create “cracks” for plasmid cloning vectors to enter. Most won’t take in the plasmid!
What are competence cells?
Bacteria cells that DO happen to take in the plasmid after transformation
When would we use DNA cloning?
Anytime we want to express a specific gene
Why is the origin of replication (ori) on a plasmid so important?
Without ori, plasmids would not be replicated by DNA polymerase because there would be nowhere to bind!
Why is the bacterial promoter on a plasmid so important?
Without a promoter, no genes would be expressed by RNA polymerase because there would be nowhere to bind!
What is an operator on a plasmid?
An operator is a segment of DNA that allows for the repressor to bind
What is a repressor on a plasmid?
Protein that binds to the operator region of DNA and blocks transcription by regulating the promoter region.
What is the polylinker region on a plasmid?
Multiple cloning site where restriction enzymes can cut to insert a gene of interest
What is the selectable genetic marker and why is it important?
Segment of DNA that codes for a protein that makes the bacterial cell resistant to antibiotics. Helps us identify the cells that have successfully taken up the gene of interest
Why is it important to be careful with which restriction endonucleases we use to insert a gene?
Genes need to be inserted in the correct direction!!
**WHY?
What is the purpose of DNA ligase?
DNA ligase comes in to seal the nick in the backbone of DNA (hydrogen bonds between bases happen spontaneously, but backbone needs to be “stitched together”)
How is a fusion protein made? Why are they useful?
CONNECTING GENE OF INTEREST WITH GREEN FLOURESCENCE
We can put the DNA of our protein of interest right next to the sequence that codes for GFP to be able to visualize a protein inside of a cell.
What is the yeast two-hybrid assay?
A way to rapidly screen for unknown proteins that interact with a particular protein of interest
- if 2 proteins interact with each other
** LOOK AT SLIDES!
What is the goal of PCR?
Allows us to detect specific DNA sequences and to amplify them so that we have enough to replicate
What does PCR require?
Thermostable (Taq) Polymerase (reads DNA)
Primers (anneal to strands)
Nucleotides
Appropriate pH
Divalent Cations (ex: Mg2+-)
- to neutralize charges
DNA Template
What are the 3 steps of PCR?
- Denaturing
(95* C to separate strands) - Annealing
(55* C to allow primers to bind template) - Extension
(72* C to synthesize new strands)
What are DNA primers?
Super short nucleic acid sequences that provides a starting point for DNA synthesis
** STUDY HOW TO DETERMINE SEQUENCES CONSIDERING 5’ AND 3’
What is the role of ddNTP in sanger sequencing?
ddNTP terminates replication (always the last base added (missing a 3’ hydroxyl group))
** LOOK UP VIDEOS
How does reverse transcription work?
Using the enzyme reverse transcriptase (isolated from retroviruses) to convert RNA to cDNA
What is cDNA? Why is it necessary?
** STUDY COMPLEMENTARY DNA
RNA is super fragile
Why make cDNA? Why not just use DNA?
cDNA is created AFTER SPLICING is complete, dramatically reducing the length of
Why is alternative splicing so useful?
Allows for multiple proteins to be coded from one gene (certain exons may be skipped in
What is a Microarray?
How do Microarray’s use cDNA?
COMPARING AND CONTRASTING GENE EXPRESSION IN 2 DIFFERENT CELLS
tens of thousands of wells–each one contains one type of probe for one specific gene, allowing for the cDNA to bind to that specific probe
Green: higher expression of sample 1cells
Red: higher expression of sample 2 cells
Yellow: expressed evenly in both kinds of cells (unchanged expression)
What is CRISPR?
A type of primitive bacterial immune system
- captures, remembers, and expresses parts of invading DNA
* LOOK AT SLIDES!
USES INVADING DNA AS A TEMPLATE TO CREATE:
* Guide RNA, which binds to the sequence of interest and activates the:
* Cas9 Protein Complex, which modifies or alters the sequence of interest
(Clustered Regularly Interspaced Short Palindromic Repeats)
What is sequence assembly? Why is it necessary?
“Shotgun Sequencing”
- Necessary because sequencing without cutting takes forever and is really expensive
What are reads?
Short sections of DNA that are cut and sequenced or lined up
What is a contig?
The label that we
What is a consensus sequence?
The actual sequence that we conclude is the real one
What is the difference between read coverage and fold coverage?
Read coverage is an average and fold coverage is the vertical coverage of a single base
What are SNPs?
What are haplotypes?
SNPs are locations where DNA can vary (one base pair)
Haplotypes are groups of SNPs that are generally inherited together
What are the general trends governing viral genomes?
If the genome is double-stranded (DNA or RNA), then it will be used directly to transcribe (+)mRNA
If the genome is single-stranded, it will be made double-stranded and then be transcribed
(ssRNA -> dsRNA)
(ssDNA -> dsDNA)
Retroviruses use reverse transcriptase to turn their RNA genome into dsDNA
(ssRNA -> cDNA
cDNA -> dsDNA
dsDNA -> integrated into genome of host cell using integrase)
* Retroviruses CANNOT do protein synthesis until their genome is integrated into the host cell
Is DNA more or less strained if it’s coiled?
DNA naturally wants to be coiled a certain amount (one turn every 10.5 base pairs). Oftentimes supercoiling is the most relaxed state for DNA, but this can also cause strain.
- Undercoiling leads to negative supercoils
- Overcoiling leads to positive supercoiling
How does RNA polymerase play a role in coiling?
The DNA ahead of polymerase will be overcoiled and the DNA behind the polymerase will be undercoiled.
What is the difference between twist and writhe?
Twist: DNA turning more or less times
Writhe: entirely new loops created by DNA helixc
What is the difference between Type I and Type II Topoisomerases?
Type I change the Lk by 1
(by cutting 1 strand)
Type II change the Lk by 2
(by cutting 2 strands)
Eukaryotic Topoisomerases
Type I
- relaxes negative supercoils
Type II
- relaxes positive or negative supercoils
- requires ATP
Prokaryotic Topoisomerases
Type I
- relaxes negative supercoils
GNA Gyrase (Type II)
- introduces negative supercoils (increases strain)
- requires ATP
G1
After mitosis, long linear chromosomes. Towards the end of G1, cohesins are added
Purpose of cohesins
rubber bands that keep sister chromatids together
G2
condenisins are added
Purpose of condensins
condense chromosomes starting at prophase and through metaphase
Metaphase
separase is added
Purpose of separase
cuts off cohesins right before anaphase
Histone subunits
H2a, H2b, H3, H4
(H1 “cinches”)
HATs
Acetylate
(activates gene expression by making DNA accessible)
HDACs
Deacetylation
(deactivates gene expression)
purpose of methylation
+CH3
keeps from acetylation
-> low gene expression
Bromodomains recognize acetyl groups and bind in order to keep nucleosomes separated
STABILIZED OPEN STATE
Chromodomains recognize
- LOOK UP IN SLIDES!
STABILIZED CLOSED STATE
What is the idea/theory behind the histone code?
Maybe we could read histone modifications (and understand modifications to histone tails) to be able to use this code to decipher whether genes will be expressed or not
cis modifications
opening or closing the dna
trans modifications
require other proteins to come in and do the change
histone modifications can be passed on to
What is the difference between the positive-sense and negative-sense strands of DNA?
Positive-Sense Strand:
NONTEMPLATE / CODING STRAND
(identical to mRNA strand after transcription)
Negative-Sense Strand:
TEMPLATE / NONCODING STRAND
(serves as template for the creation of mRNA)
A->T
T->U
C->G
G->C
How do we determine two primer sequences from one strand of DNA?
5’ —————- 3’ (+) DNA
3’ ——– 5’ (-) Primer
5’ ——– 3’ (+) Primer
3’ —————- 5’ (-) DNA
What does sgRNA stand for? What is its purpose in the CRISPR/Cas9 nuclease system?
Single Guide RNA pairs the CRISPR/Cas9 to a specific location on the target DNA and activates the nuclease in order to cut and modify that sequence.
How do we determine which restriction endonucleases to use in order to move a gene of interest from one plasmid to another?
PAY ATTENTION TO DIRECTIONALITY!
The order of cuts by endonucleases must match so that genes are cut and inserted in the right direction
What is the difference between genomics and linkage analysis?
Genomics is the study of an organism’s entire genome, which includes all of its genes and their interactions.
Linkage analysis is a method used to map the location of genes on chromosomes and determine the association between genetic markers and inherited traits.
Why wouldn’t DNA Polymerase be used to replicate hundreds of telomeres lined up with the sequence TTTAGGG?
What is used instead?
DNA Polymerase has a tendency to fall off of repeated sequences or to slip and skip stuff.
- TELOMERASE IS USED INSTEAD
