New Lectures for the Final Flashcards
(123 cards)
1
Q
What are restriction enzymes?
A
Enzymes that recognizes a specific sequence of bases anywhere within the genome and cuts sugar-phosphate backbones of both strands
2
Q
What are restriction sites?
A
- sequences recognized by restriction enzymes
- usually 4 – 8 bp of double-strand DNA
- Often palindromic – base sequences of each strand are identical when read 5’-to-3’
3
Q
What are blunt ends vs. sticky ends?
A
• Blunt ends – cuts are straight through both DNA strands at the line of symmetry • Sticky ends – cuts are displaced equally on either side of line of symmetry – Ends have either 5' overhangs or 3' overhangs
4
Q
How is the length of fragments generated by restriction enzymes calculated?
A
General formula for fragment length is 4^n, where n is the number of bases in the recognition site
5
Q
What are some mechanical forces that can create DNA fragments?
A
– Passing DNA through a thin needle at high pressure
– Sonication (ultrasound energy)
6
Q
What information does DNA Gel Electrophoresis provide?
A
- relative size of DNA fragments
7
Q
What are the three main features of plasmid cloning vectors?
A
– Origin of replication – A selectable marker gene (for example antibiotic resistance) – A synthetic polylinker, DNA sequence containing multiple restriction enzyme sites
8
Q
How is DNA inserted into plasmid vectors?
A
Digestion of the vector and human genomic DNA with a restriction enzyme results in complementary sticky ends that are put together by DNA ligase
9
Q
What is Sanger Sequencing?
A
Gene sequencing technology
10
Q
What materials are needed for sanger sequencing?
A
- single-stranded DNA fragments
- hybridized templates and primers
- DNA polymerase
- dNTPs
- ddNTPs (unique fluorescent tags attached)
11
Q
What is different about ddNTPs from dNTPs?
A
- ddNTPs lack a 3’ -OH
- halts polymerization
12
Q
What are the results of Sanger Sequencing?
A
- DNA fragments separated by gel electrophoresis
- Each DNA fragment is tagged at the 3’ end with a ddNTP attached to unique fluorochrome
- Gel is read by lasers and a computer
- computer puts together DNA sequence based on fluorophores from different length fragments
13
Q
What is a BAC?
A
- Bacterial artificial chromosomes
- alternative cloning vector
- carries large inserts
14
Q
What is the Shotgun strategy?
A
The shotgun strategy takes DNA and breaks it up into fragments that are then constructed into a BAC library. A computer then sequences all the fragments of DNA and constructs an entire genome based on overlapping sequences (contigs)
15
Q
Why does a BAC clone give you two sequence reads rather than one? (Paired-end sequencing)
A
DNA inserts can be too long to sequence so ~1000 bp sequences can be read from both sides of the insert starting at the first and second primer. This also lets you know that these two sequences are ~200-300 kb apart. The read can be done again starting at the end of the two sequences that were learned before until you have reached the overlapping sequence from both ends.
16
Q
Why do repeat sequences prevent correct assembly of single shotgun sequence reads?
A
The computer is putting together DNA fragments like they are a puzzle. Repeat regions make it impossible for the computer to differentiate certain puzzle pieces (DNA fragments), meaning there is a possibility it is put together incorrectly
17
Q
How are cDNA libraries made?
A
- mRNA is taken from red blood cell precursors
- Add DNA dinucleotide primer
- treat with reverse transcriptase in the presence of other dinucleotides
- denature mRNA/cDNA hybrid and digest mRNA with RNAse
- 3’ end of cDNA folds back on itself to act as a primer
- The first cDNA strand acts as a template for the synthesis of the second DNA strand with DNA polymerase
- results in cDNA double helix that can be inserted into a vector
18
Q
How are cDNA libraries different from genomic libraries?
A
cDNA libraries only contain sequences from exons while genomic libraries contain the entire genome sequence
19
Q
why are cDNA levels different in different parts of the body?
A
different genes are expressed more or less in different cell. (e.g. brain cells will express different mRNAs than liver cells)
20
Q
What is the largest DNA fragment that a plasmid can accommodate?
A
tops out at 20kb
21
Q
What is an open reading-frame? (ORF)?
A
a reading-frame uninterrupted by stop codons
22
Q
how long does a stretch of DNA with no stop codon need to be to indicate there is likely an open reading frame there?
A
4 bases and 3 bp in a codon –> 4^3 = 64 different codons
3 possible reading frames/strand -> 64/3 = 21 aa
If a frame codes for more than 21 amino acids with no stop codon, it is indicative of an open reading frame
23
Q
How much of the genome is conserved between species in protein coding regions compared to non-coding regions?
A
Protein coding regions have a much higher percentage of conservation between species than the entire genome at large
24
Q
What are examples of non-coding RNAs (ncRNAs)?
A
rRNAs, tRNAs, and snRNAs (small nuclear RNAs)
25
How are mRNAs sorted from other RNAs in eukaryotic cells?
PolyA tails can be hybridized to oligo-dT (single strand DNA fragments of 20
nucleotides made of dT only) that can be tagged for identification
26
Why are cDNAs sometimes misleading when it comes to determining the sequence of the gene in the genome?
```
- Alternative splicing means a
single gene can produce
different proteins,
complicating the prediction
of the proteome (all
proteins made in an
organism)
- Important to sequence many
individual cDNA clones from
libraries made using mRNAs
from different tissues
```
27
What is an exome?
The part of the genome corresponding to exons
28
How much of the genome is made up of exomes?
• Exome = 1.5−2%
• Remainder is introns, centromeres, telomeres, transposable elements, etc.
• Variation in genome size mostly due to changes in noncoding DNA rather than gene
number or size
29
What are the two types of repetitive DNA?
- multicopy tandem repeats
| - transposable elements
30
what is junk DNA?
Repetitive DNA with no known function
31
What are gene-rich regions?
• Chromosomal regions that have many more genes than expected from average gene density over entire genome
• Example in human genome –class III region of major
histocompatibility complex
32
What are gene deserts?
* Regions that have no identifiable genes
* Largest is 5.1 Mb on chromosome 5 with no identified genes
* Biological significance of gene-rich regions and gene deserts is not known
33
What is the most gene rich region of the human genome?
- Class III region of the human major histocompatibility (MHC) complex
- MHC complex contains 60 genes within a 700 kb region
34
What are domain architectures?
- different numbers and kinds of protein domains in unique orders
- Shuffling, addition, or deletion of exons during evolution can create new domain architectures
35
What is a homeodomain consensus sequence?
Function of new protein can be deduced if it contains a domain
known to play a role in other proteins
36
What is exon shuffling?
Exon shuffling is a molecular mechanism for the formation of new genes. It is a process through which two or more exons from different genes can be brought together ectopically, or the same exon can be duplicated, to create a new exon-intron structure.
37
What are gene families?
Gene families are groups genes closely related in sequence and
function
38
How do duplications and divergence of genes create gene families?
```
Duplicated DNA
sequence products
start out identical,
eventually diverge
via accumulation of
mutations that eventually lead to new genes with closely related functions
```
39
what are orthologous genes?
arose from the same gene in the common ancestor, usually retain
same function
40
What are paralogous genes?
arise by duplication, often refers to members of a gene
| family
41
What is Homology
blanket term for all evolutionarily related sequences
42
What are Pseudogenes?
sequences that look like, but do not function as, genes
| • Rapidly accumulate mutations
43
What are de novo genes?
genes without homologs
| • Young genes that evolved recently from ancestral intergenic sequences
44
What are Syntenic blocks?
homologous blocks of
chromosomal sequence
• Mouse and human genomes diverged 85 million years ago, but
can be compared via chromosomes to visualize similarities
45
How does Combinatorial amplification results in greater complexity from fewer genes?
* Example – human T-cell receptor family
* DNA rearrangement combines V, D, and J segments into a gene
* Result is about 1000 different combinations
* 45 V X 2D X 11J = 990 X 2C = 1980 combinations from 60 elements
46
How many bp can nanopore sequence sequentially?
NANOPORE TECHNOLOGY allows sequence reads of 1,000,000 bp with modest accuracy
47
What are DNA polymorphisms?
sequence differences
48
Why is there no wild-type human genome?
- Too much variation
- The genome sequences of only three people reveal over 5 million
DNA polymorphisms
49
What are the 4 categories of genetic variation?
- Single nucleotide polymorphism (SNP)
- Insertion/Deletion (DIP or InDel)
- Simple sequence repeat (SSR)
- Copy number variant (CNV)
50
What produces copy number variant?
• Unequal crossing-over produces new alleles of copy number variants (CNVs)
• Misalignment during meiosis
leads to unequal crossing over.
• Not a common event, so most
CNVs are inherited, rather than being a new mutation.
51
How are single-base mismatches distinguished?
• Hybridization of short (< 40 bases) oligonucleotides to sample (target) DNAs
(allele-specific hybridization)
• If there is no mismatch between probe and target, hybrid will be stable at high temperature
• If there is a mismatch between probe and target, hybrid will not be stable at high temperature
52
How are microarrays used for genotyping?
- Allele-specific oligonucleotides (ASOs) are attached to a solid
support (like a silicon chip)
- DNA is fragmented, ligated to adapters, amplified, denatured, and coupled to a fluorescent dye
- DNA is added to microarray and anneals to oligonucleotides with complimentary bases
- fluorescent output is proportional to the number of copies of each allele
53
How are microsatellites formed?
As polymerase is sequencing new DNA, it pauses. At the leading end of the new strand, double helix "unzips" a bit. On occasion, these strands reanneal out of place, and polymerization continues.
54
How are SSR and Microsatellite repeats detected?
- the unique DNA that flanks the microsatellite or SSR is used to make PCR primers
- PCR is run and alleles with different number of repeats can be separated on gel
55
What are copy number variations (CNVs)?
CNVs are tandem sequence repeats more than 10 bp long
56
What are the steps involved in positional cloning?
• Region of interest narrowed by finding closely linked DNA markers.
• Candidate genes are located in the region of interest.
• Sequence and expression of
candidate genes are determined
in normal and diseased individuals.
• difference in sequence correlates with phenotype
57
What are Log of Odds (LOD) Scores measuring?
How much more likely is it that the allele transmission pattern seen in a pedigree will occur if
the loci are linked at the observed frequency than if they are not linked.
58
what is step 1 of NGS?
sample prep (attach linkers to DNA fragments)
59
what is step 2 of NGS?
Cluster generation (formation of hooped DNA on the Illumina flow cell)
60
what is step 3 of NGS?
Sequencing by synthesis (feeding polymerase one fluorescent nucleotide base at a time and using optical methods to trach each incorporated base)
61
What is step 4 of NGS?
Data analysis (either assembly of a genome from overlapping parts or using the sequences directly in blast to perform a 16S microbiome analysis)
62
What is positional cloning?
Is identifying the POSITION of a given gene
63
How are linkage maps created?
- 2 point, 3 point mapping
- gels
- ASO chips (allele specific oligonucleotides)
- sequencing
64
What are some limitations to Positional cloning?
- crosses with two heterozygous parents wont be informative with heterozygous offspring
- linkage map requires at least one parent to be double heterozygote
- small families and few pedigrees with provide incomplete data
65
What is the equation for LOD?
LOD = log10[P(L)/P(NL)]
```
P(L) = probability of linkage
P(NL) = probability of no linkage
Odds = P(L)/P(NL)
```
LOD must be >3 to conclude genes are linked
66
What are the 4 types of chromosomal rearrangements?
- deletions
- duplications
- inversions
- reciprocal translocations
67
What causes chromosomal rearrangement to occur?
- Double strand breaks followed by nonhomologous end-joining
- Crossing over between repeated
sequences on homologous or
nonhomologous chromosomes (Aberrant crossing-over)
68
How does crossover between repeats create each type of chromosomal rearrangement?
• in same direction on same
chromosome → deletion
• In opposite orientation on same
chromosome → inversion
• on misaligned homologous chromatid or chromosome → Dp and Del (∆)
• On nonhomologous chromosomes → reciprocal translocation
69
How de deletions affect recombination?
```
• No recombination can
occur within a deletion loop
• Consequently, genetic map
distances in deletion
heterozygotes will not be
accurate
```
70
How can deletions be used to map genes?
```
•Examine phenotype of a
heterozygote for recessive
allele and deletion:
•If the phenotype is
mutant, the mutant gene
must lie inside the deleted
region
•If the phenotype is wildtype, the mutant gene
must lie outside the
deleted region
```
71
Why are duplications important for evolution?
new functions arise as a
process of duplication of genes followed by divergence of the expression or function of the gene product
HOWEVER
very large duplications are deleterious (gene dosage
problem)
72
What are the different types of duplications?
Tandem duplications
- same order (BCBC)
- reverse order (BCCB)
Nontandem (dispersed) duplications
- same order (ABCDEFBCG)
- reverse order (ABCDEFCBG)
73
What are the phenotypic effects of duplications?
• Most duplications have no phenotypic consequences;
• Novel phenotypes may occur because of increased gene copy number or because of
altered expression in new chromosomal environment
• Homozygosity or heterozygosity for a duplication can be lethal or harmful
- Depends on size of
duplication and affected
genes
74
What are the types of inversions?
```
• Pericentric inversion –
centromere is within the
inverted segment
• Paracentric inversion –
centromere is not within
the inverted segment
```
75
How do inversions affect crossovers during recombination?
```
• Homologous regions in
inverted chromosomes can
still pair and undergo
crossover
• Crossing over within the
inversion loop produces
aberrant recombinant
chromatids
- non-viable zygotes
produced
```
76
What is the result of Pericentric
| inversion crossover?
Each recombinant chromatid has a centromere, but each will
| be genetically unbalanced (nonviable)
77
What is the result of Paracentric
| inversion crossover?
One recombinant chromatid
lacks a centromere and the
other recombinant chromatid has two centromeres (dicentric chromatid)
Dicentric chromatid breaks randomly (nonviable)
78
What are reciprocal translocations?
Translocations attach part of one
chromosome to a nonhomologous
chromosome
79
How do reciprocal translocations affect the genotype and phenotype?
* If the breakpoints of a reciprocal translocation do not affect gene function, there are no genetic consequences in homozygotes
* No loss of genetic material
* Usually don’t result in mutant phenotype
* May result in mutant phenotype if breakpoint is within or near a gene.
* May result in decreased fertility
80
How do reciprocal translocations affect chromosome segregation?
Three chromosome segregation patterns are possible in a translocation heterozygote
• Balanced gametes are produced only by alternate segregation,
and not by adjacent-1 or adjacent-2 segregation
81
Why do heterozygotes with reciprocal translocations display pseudolinkage?
In a reciprocal translocation heterozygote, only the alternate segregation pattern results in viable progeny
• In outcrosses, genes located on the nonhomologous chromosomes would behave as if they are linked
82
What are Robertsonian translocations?
- Robertsonian translocations can reshape genomes
- Arise from breaks at or near centromeres of two acrocentric
chromosomes. (having the centromere situated so that one chromosomal arm is much shorter than the other.)
- The small chromosome may be lost from the organism.
83
What are transposable elements?
• Transposable elements (TEs): small virus-like DNA elements
that can move from one genomic location to another
• A TE can be a mutagen: it can hop into a gene and inactivate it
• TE movement can generate chromosomal rearrangements
84
How can recombination in region of homology in reciprocal translocation heterozygote help calculate gene map distances?
recombinant/ total = map distance of gene from translocation breakpoint
85
What steps of gene expression are involved in regulation?
* Transcription initiation
* Transcript processing
* Export from nucleus
* Translation of mRNA
* Protein localization
* Protein modification
86
What are major cis-acting regulatory elements?
- promoters
| - enhancers
87
What are promoters?
* DNA sequence that is usually directly adjacent to the gene
* Bind RNA polymerase
* Often have TATA box:
* Allow basal level of transcription
88
What are enhancers?
* DNA sequence that can be far away from gene
* Augment or repress the basal level of transcription
* May be located either 5’ or 3’ to the transcription start site
* Still function when moved to different positions relative to promoter
89
How are enhancers identified?
• Constructing a recombinant DNA molecule that has a putative enhancer sequence fused to a reporter gene such as the green fluorescent protein
(GFP)
• Generating a transgenic organism that has the recombinant DNA in its genome.
• test levels of GFP in organism without enhancer versus GFP levels of organism with enhancer
90
What are trans-acting regulatory elements? (Transcription factors)
- Proteins act in trans to control transcription initiation
- Sequence specific DNA binding proteins
- Bind to promoters and enhancers
- Recruit other proteins to influence transcription
- Three types: basal factors, activators, repressors
91
What are basal factors?
- class of proteins that bind to specific sites (promoter) on DNA to activate transcription
- Basal factors bind to promoters of protein-encoding genes
```
Ordered pathway of
assembly at promoter:
1. TBP binds to TATA box
2. TAFs bind to TBP
3. RNA pol II binds to TAFs
```
92
What is a mediator complex?
- a complex of more than 20 proteins
- bridge RNA pol II at the promoter and activator or repressor proteins at the enhancer
- doesn't bind DNA directly
93
What are activators?
activators are proteins that bind the enhancer region of DNA and recruit RNA pol II complex to basal promoter
94
What are coactivators?
- proteins that are recruited by the activator protein
| - displace nucleosomes to open chromatin structure for transcription
95
What are the functional domains of activator proteins?
* DNA binding domain: binds to specific enhancer
* Activation domain: binds to other proteins (basal factors or coactivators)
* Dimerization domain: SOME activators also have a domain that allows them to interact with other proteins
96
Homodimers vs. Heterodimers
```
• Homodimers: multimeric
proteins made of identical
subunits
• Heterodimers: multimeric
proteins made of nonidentical subunits
```
97
What are repressor proteins?
- prevent transcription of DNA
98
How do repressor proteins work?
- recruit corepressors that directly prevent RNA pol II complex from binding promotor
99
What are the two functions of corepressors?
* Prevent RNA pol II complex from binding the promoter
| * Modify histones to close chromatin structure
100
What are indirect repressors?
- proteins that interfere with the function of an activator
101
What are the different functions of an indirect repressor?
* Competition due to overlapping binding sites
* Repressor binds to activation domain (quenching)
* Binding to activator and keeping it in cytoplasm
* Binding to activator and preventing homodimerization
102
How are transcription factors identified?
by fusing GFP to promoter region of gene and observing the effects of GFP expression in organisms with mutations in activator and repressor genes
103
What is the Max network?
Family of related “basic helix-loop-helix” DNA binding proteins that bind DNA as dimers
- Family is Max, Myc, and Mad proteins
104
What is the Max protein?
- Max protein is a member of the Max network.
- present in all cells
- can form homodimers or heterodimers with other family members (Mad and Myc)
- other family members can only form heterodimers with Max (they are present at different levels in different cell types)
105
What is the Myc protein?
- Activator protein in the Max network
| - can only form dimers with Max
106
What is the Mad protein?
- repressor protein in the Max network
| - can only form dimers with Max
107
How is Cell-type specific transcription is achieved
by changes in transcription factors
• Allosteric interactions (ex. steroid hormone receptor binds to enhancer only when bound to steroid hormone)
• Modification of transcription factors (ex. phosphorylation)
• Transcription factor cascades
108
How is gene expression regulated by hormones?
* rapid
* NR (nuclear receptor) is sequestered in the cytoplasm
* Binding of membrane permeable steroid hormone to the NR causes a conformational change and forms dimers
* NR-hormone complex translocates into the nucleus where it binds to Hormone Receptor enhancer elements
* Recruits coactivators and basal factors
109
What is Chromatin immunoprecipitation-sequencing (ChIP-Seq)?
tool for finding all target genes of a particular transcription factor
within the entire genome of a particular type of cell
110
what are the steps of Chromatin immunoprecipitation-sequencing (ChIP-Seq)?
* Crosslink DNA and protein component of chromatin
* Fragment DNA
* Allow an antibody specific to the protein of interest to bind
* Purify complexes with antibody, protein of interest and DNA fragments
* Sequence DNA
111
How does an enhancer know which genes to regulate?
Insulator sequences are located between enhancers and unrelated promoters. These sequences prevent the enhancer from influencing the unrelated gene
112
How does an insulator sequence function?
- Human insulators bind CTCF proteins to form loops called
topologically associating domains (TADs)
- enhancers will only be able to activate promoters that are located on the same loop
113
How is mRNA translation regulated in response to nutrients?
* 4E-BP1 binds to initiation factor eIF4E, blocks initiation
* Presence of nutrients and growth factors in the environment leads to phosphorylation of 4E-BP1
* Once phosphorylated, 4E-BP1 inactivates and initiation can begin
114
Which enzymes are involved in translational control through polyA tail length?
- Deadenylase: polyA tail is shorten -> less translation
| - Poly-A polymerase: polyA tail is lengthened -> more translation
115
What are the three types of small RNAs and how do they regulate mRNA translation?
1. miRNAs
2. siRNAs
3. piRNAs
Specialized RNAs that prevent expression of specific genes through complementary base pairing; 21-30 nt long
116
what is the structure of primary transcripts of miRNAs?
The primary transcripts have double-stranded stem loops
117
What are the three proteins required for processing miRNA?
- Drosha
- Dicer
- RISC (miRNA-induced silencing complex)
118
What is the purpose of Drosha?
excises stem loop from primary miRNA (pri-miRNA) to generate pre-miRNA
119
What is the purpose of Dicer?
process pre-miRNA to a mature duplex miRNA
120
What are the two ways in which miRNAs can down-regulate expression of target genes?
1. when complementarity is perfect, target mRNA is degraded
| 2. when complementarity is imperfect, translation of mRNA target is repressed
121
How are siRNAs (small interfering RNAs) produced?
• sources of dsRNA are precursors of siRNAs
- transcription of both strands of endogenous genomic sequence
-arise from exogenous virus
• dsRNAs are processed by Dicer
122
How do siRNAs function?
* Form ribonucleoprotein complexes with Argonaute proteins
| * Interfere with gene expression or may destroy viral mRNAs
123
How do piRNAs function?
* minimize transposable element mobilization
* make complexes with Piwi proteins
* Complexes modify histones to interfere with TE transcription or degrade TE RNAs
