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Flashcards in Test 3 Deck (211):
1

Need for PCR

-DNA template
-Primers
-DNTPs
-Taq polymerase

2

Need for sequencing (Sanger)

-DNA template
-1 primer
-DNTPS
-Taq polymerase
-ddNTPs

3

Why can't new nucleotides bind to ddNTP

no OH, just H

4

ddNTP

Terminates synthesis

5

dNTP

Extends DNA strand

6

PCR

Laboratory tecnique used to make millions of copies of a particular region of DNA

7

Process of sequencing

Primer attaches to unique sequence of single stranded DNA template
New nucleotides added until ddNTP added at random

8

ddATP

Fragment will end in A

9

Poly a gel

Used for sequencing

10

What is the smallest fragment on the poly a gel

Primer

11

How to read sequence in poly a gel

Bottom to top

12

Transformation

Stress cells so it opens pores and takes up foreign DNA (plasmids inserted)

13

Clone bank

Bacteria with templates that represent entire genome

14

Problems with PCR

-Error prone (dont want to risk mutations. Living system has proofreading)
-Protects integrity of DNA
-Only small volume used in PCR (if you transfer bacteria to a new plate, trillions more will grow)

15

Plasmids

Nonchromosomal DNA molecules in bacteria

16

What are plasmids designed to do

Carry a gene of interest to be inserted

17

What must happen to the plasmid to insert the gene

Must be cleaved

18

What cleaves the plasmid

Restriction enzymes

19

What is a vector

What DNA enters the host organism through (plasmid)

20

How do restriction enzymes know where to cut

They recognize a specific short DNA sequence (when this sequence is found, it is cleaved)

21

Characteristic of restriction sequences

Palandromic. Same sequences found 5-3 on both strands

22

Blunt end

Cut site between 2 nucleotides. No extra nucleotides sticking out (straight through strands or at lines of symmetry)

23

Overhang (sticky ends)

Cut site leaves nucleotides sticking out (3' or 5' overhang possible)

24

Polylinker

Plasmid with multiple cut sites (has unique restriction sites that dont occur anywhere else in the plasmid)

25

How is the DNA that is to be inserted cut and why

With same restriction enzyme so it ends up with the same overhangs as the plasmid

26

What is needed for insertion of gene

Vector
Target fragment
Ligase to seal target fragment into vector

27

Competant bacterium

One capable of transformation

28

Restriction enzyme nomenclature

EX: EcoRI
E= genus
co=species
R=strain
I=sequence number from which it was isolated

29

Chances of a certain 4 base sequence occurring

(1/4)x(1/4)x(1/4)x(1/4)

30

Breaking DNA

Needle
Pipette
Sonication

31

What does breaking DNA cause

Long random overhangs

32

What is needed for plasmid cloning vectors

-Origin of replication
-Selectable marker
-Restriction sites for cloning insert DNA

33

What does the selectable marker do

Gets rid of bacteria not transformed

34

What will a plasmid sometimes do

Self anneal withot the gene

35

How to determine number of clones in a perfect library

Length of genome divided by average size of inserts

36

Approaches of sequencing

Shotgun approach and ordered approach

37

Shot gun approach

DNA randomly sheared, computer finds overlapping sequences

38

Ordered approach

DNA cut into large pieces, figures out where they belong and then cuts into smaller pieces

39

Restriction map

Determines what original strand looked like

40

How is sequencing done in eukaryotes

mRNA is sequenced (no introns), converted back to DNA using reverse transcriptase (cDNA)

41

Single spike

Both alleles have the same nucleotide

42

2 spikes

Different nucleotides

43

VNTR

Varied number of tandem repeats

44

Multiplexing

Multiple primer pairs in a single reaction tube (each labelled with a different flourescent molecule)
-up to 26 bands, few as 13

45

How is DNA probe produced

Someones DNA
-complements DNA on microarray

46

What happens to the probe if it is not a perfect complement to the DNA in the microarray

Probe is removed when heated

47

What does the probe determine

Which part of the microarray could be hybridized. Each has a particular allele type (EX: levels of expression in breast cancer)

48

Preparing DNA for hyridization

1. Fragmented
2. Adapter attached (sequence we have attached)
3. Amplified by PCR and denatured to make single stranded
4. Flourescent molecule attached for detection later on

49

Allelic heterogeneity

Disease caused by different mutations in the same gene

50

Compound heterozygote

Different mutant alleles of same gene

51

Locus heterogenity

One gene of the 2 has mutation

52

Karyotype

Flourescent probes let you see changes in chromosome (parts missing)

53

Deteltion

Loss of genes

54

Duplication

Multiple copies of a gene

55

Inversion

Location of gene switched

56

Translocation

Chromosomes swap information

57

Why does translocation location matter

If 2 centromeres on one chromosome, the organism will not be viable

58

Double strand breaks in haploid cell

NHEJ

59

Consequences of NHEJ

Pieces can be lost, duplicated, inverted, translocated

60

What happens when homologs break

Duplication and deletion

61

What happens when nonhomologs break

Translocation

62

Homologous recombination errors

-loop formed and removed (deletion)
-Inversion can occur if within circle

63

What can be used to detect breakpoints

PCR

64

How to tell if there was a deletion based on PCR

If product is smaller than expected

65

Position of primers in PCR

Face eachother and on opposite strands

66

What happens if the primers are facing away from eachother

No product is formed

67

Deletion heterozygote

One allele is normal, the other is deletion

68

What is formed in recombination when there was a deletion

Deletion loop

69

What affect does the deletion loop have

Does not have a complement
Brings genes that are normally far apart closer together

70

How can evolution occur

Altered gene copy number passed on to offspring. Mutations build up as consequence, can take on new function (evolution)

71

Tandem duplication

Duplication in series on the same chromosome

72

Nontandem

Duplications far apart

73

Inversions dont affect the phenotype unless...

Gene is broken or if in the germ line

74

What is a recombination and crossover suppressor (no homology)

Inversions

75

Pericentric

Inversion includes centromere (included in loop)

76

Paracentric

Inversion doesnt include centromere

77

Reciprocal translocation

No loss or gain of genetic material

78

Effect of reciprocal translocation

Normal in somatic cells, decreased fertility in germ cells

79

Transposable elements

Segment of DNA that evolves ability to move place to place within a genome

80

3 transposable elements

LINEs, SINEs, HERVs

81

What do LINE SINE and HERV contain

Reverse transcriptase and RNA binding protein

82

Reverse transcriptase

Turns it back to DNA so it can insert itself back into genome

83

SINEs

Portions of LINEs. Not able to copy themselves

84

HERVs

Retroviral transposons. Have terminal repeats. Have endonuclease activity (cuts 5 base sequences and inserts cDNA into cut location)

85

What is different between SINE, LINE, HERV, and DNA transposons

DNA transposons have no RNA intermediate, go straight into the genome

86

DNA transposons

-transposase gene make protein to bind and help copy sequence to another location
-Only some carry gene but all carry sequence

87

What is the problem with large transposable elements

Can interfere with crossing over (chromosomal abnormalities)

88

What happens if transposase only binds to end IR sequences

Transposition occurs

89

What genes are commonly found on transposable elements

Antibiotic resistance genes

90

How are antibiotic genes transferred from chromosomal dna

Transferred to plasmid. Plasmids can be trandferred to other organisms

91

Aneuploidy

Missing or extra homolog

92

Nullisomy

Whole chromosome pair missing (22 chromosomes)

93

Euploidy

Normal

94

How is aneuploidy caused

Nondisjunction during meiosis I or II (anaphase) or mitosis (same frequency as meiosis II)

95

What organisms is polyploidy normal in

plants (lethal in humans)

96

Polyploidy

Greater than 2n

97

Colchisine

Prevents formation of spindle fibers, abnormal number of chromosome following each cycle. Cell will not undergo cytokinesis (triploid cells)

98

What happens when a triploid cell divides

Chromosome number in daughter cells will be unbalanced (sterility)

99

Alloploid

Hybrid formed from distinct but related species. Chromosome sets cannot pair easily. Results in sterile organism

100

Amphidiploid

Hybrid of 2 diploid genomes from different parental species. Infertile because no homologous chromosomes

101

What are the majority of cella that make up a human

Bacteria

102

What do bacteria do on the skin that is beneficial

Take up available binding sites on the skin so harmful bacteria cannot colonize

103

Characteristics of bacteria

- No nuclear membrane (nucleoid only)
-Free ribosomes
-No membrane bound organelles
-Most have cell wall
-Pili on outside
-flagellum

104

Pilli role

Attachment and gliding

105

Flagellum role

Movement through liquid

106

Pathogenic bacteria

Invade tissues and produce toxins (tetanus)

107

How does DNA fit in a bacterial cell

Supercoiled

108

Pangenome

All of the genome plus the genes found in only some of the strains but not others

109

Core genes

Genes that all the members of a species have (usually around 1000)

110

Tn elements

Composite transposable elements

111

What types of genes do plasmids have

Never have genes required for metabolic processes. Only carry genes that benefit them

112

What type of plasmid is found in high copy numbers

Small plasmids

113

What type of plasmid is found in low copy numbers

Large plasmids

114

What ensures that a plasmid will not be lost

If it provides benefits

115

Why are there multiple copies of plasmids

Based on gene dosing. More copies = more resistance

116

What happens if a small and large plasmid carry the same gene

The small plasmid will be lost

117

r plasmid

Resistant plasmid, has mant resistant genes

118

Can genes on the plasmids be transferred to the genome

Yes because theyre on transposons

119

Metagenomics

Collective analysis of genomic DNA from an entire community of microbes (ex: pondwater)

120

Methods of growing bacteria

-Liquid medium
-agar plate

121

What to use a liquid medium for

Fast growth

122

What to use an agar plate for

Analytical purposes, not fast growth

123

How many chromosomes do all bacteria have

One (monoploid), plasmids dont count

124

Auxotroph

Needs certain things in the growth media to grow (EX: amino acids that it doesnt produce on its own)

125

Screening

Determine organisms present and their properties

126

Selection

Excludes certain bacteris or organisms (EX: select only organisms that have a certain gene)

127

Vertical gene transfer

Traits transferred from parent to offspring

128

Horizontal gene transfer

Traits introduced from unrelated individuals

129

Types of horizontal gene transfer

1. Transformation
2. Conjugation
3. Transduction

130

Process of transformation

-Bacterial DNA picked up by cells that are stressed (become competant)
-Stray DNA enters through pores
-Recombines with organism through homologous recombination

131

What is required for transformation to occur

Only works if there are homologous sequences in the genome already

132

Conjugation process

F+ has sex pilus
-One strand of DNA transferred through sex pilus to other cell. Second strand reforms in both cells
-Second cell becomes F+ once DNA gets there

133

Where are fertility genes found

Plasmid

134

Types of transduction

Generalized and specialized

135

Types of phages

Lytic and temperate

136

Generalized transduction phage

Lytic

137

Generalized transduction process

-Transfer of DNA by virus
-Genome of virus enters and replicates in the cell
-Copies of protein coat from virus made in cell
-Components come together and break up bacterial genome (some take up bacterial DNA)
-Cell will eventually lyse

138

What happens when a virus with bacterial DNA infects another bacterial cell

Bacteria has new DNA it can use

139

Artificial transformation

In the lab. Cells treated with calcium chloride to make membranes permeable to DNA

140

How to tell what cells have been transformed

Based on the media they grow in

141

Specialized transduction phage

Temperate phage

142

Specialized transduction process

-When viral DNA comes out, it can be exised incorrectly and take out a small piece of bacterial DNA with it (turns into virus and lyses cell)
-This bacterial DNA can be inserted into another bacteria by virus

143

When cell doesn't lyse (specialized)

Cell replicates viral DNA (integrated into genome)--> more cells with viral particles produced
-Viral DNA can remove itself whenever it wants and lyse the cell

144

What exactly does the restriction enzyme cute

Sugar phosphate backbone

145

How are restriction fragments generated

Digestion of DNA with restriction enzymes

146

How long are restriction sites usually

4-8 bp of double stranded DNA

147

Where does each restriction enzyme cut

Same place relative to its specific recognition sequence

148

What does the fragment length produced depend on

The restriction enzyme used

149

Average fragment length

4^n (n is number of bases at the recognition site)

150

What forces can be used to break phosphodiester bonds

Mechanical (needle, sonication, pipette)

151

Molecular cloning

Purify specific DNA fragment away from other fragments to make many identical copies of it

152

Steps to molecular cloning

1. Insert DNA fragments into cloning vectors to make recombinant DNA molecule
2. Transport recombinant DNA to living cell to be copied

153

What does digestion of the vector and human genomic DNA with a restriction enzyme result in

Overhangs (sticky ends)

154

What seals phosphodiester bonds between vector and insert

Ligase

155

Genomic equilvalent

Number of clones in a perfect library

156

What does DNA polymerase need

dNTP
Primer
Template

157

How do template and primer interact

Hybridization

158

Nested array

Fragments made by sanger sequencing. Each differs in length from preceeding and succeeding sequence by one nucleotide

159

How are different ddNTPs detected in gel

Each marked with a different flourescent molecule

160

What direction are sequences read in

5 to 3

161

What is ambiguity in sequence recorded as

N

162

Open reading frame (ORF)

Uninterrupted by stop codons

163

Why is mRNA not easily sequences

Too rare to purify and sequencing technology is not widely available

164

What is mRNA converted to for sequencing

cDNA

165

Creating complement to cDNA

mRNA digested with RNAse
-3' of cDNA folds back and acts as primer for second strand synthesis
-Polymerase adds dNTP
(can be added to plasmid)

166

Exome

Part of genome that is corresponding to exons

167

Gene rich regions

Chromosomal regions with many more genes than expected

168

Gene deserts

Areas with no identifiable genes

169

Gene families

Groups of genes closely related in sequence and function

170

Pseudogenes

Look like genes but do not function as genes

171

Orthologous genes

Arose from the same gene in common ancestor, usually retain the same function

172

Paralogous genes

Arise by duplication, often refers to members of gene family

173

Syntenic blocks

Colored segments contain at least 2 genes whos order is conserved

174

Chromosomal rearrangements

Have moved blocks to different places

175

GenBank

Database (sequencing)

176

RefSeq

Annotated version of species genome

177

DNA polymorphisms

Sequence difference vs standard human genome

178

Single nucleotide polymorphisms

One base pair changes

179

Deletion insertion polymorphisms

Short insertions or deletions of a single or a few base pairs

180

Simple sequence repeats SSR

1-10 base sequence repeated 15-100 times tandem

181

Copy number variants (CNV)

Larger blocks of duplication or deletion with population frequency of <1%

182

What produces new alleles of CNVs

Unequal crossing over

183

How to people get most CNVs

Inherited

184

Size of CNV

Repeats more than 10bp long

185

What is PCR

Method for making copies of target region of DNA

186

Steps to PCR

1. Denature
2. Base pairing of primers
3. Polymerization from primers along templates

187

Multiplexing PCR assay

More than one target sequence amplified in a single reaction tube

188

How is multicolor banding produced in karyotype

FISH probe

189

What results from homozygoty of deletions

Lethal usually (depends on size and affected genes)

190

Deletions heterozygote characteristics

Can have mutant phenotype due to gene dosage (haploinsufficiency)
-May uncover existing recessive mutant alleles

191

What can occur because of duplications

Novel phenotypes because of altered expression in chromosomal environment

192

Homozygotity and Heterozygotity in duplication

Can be lethal (depends on size and affected genes)

193

Abnormal phenotypes in inversions occur if

- Inversion disrupts a gene
-Inversion places a gene near regulatory sequences for other genes or near heterochromin

194

What does pericentric result in

Reduced fertility. Each will have centromere but be genetically imbalanced.

195

What does paracentric result in

Reduced fertility. One recom. chromatid will lack centromere and the other will have 2

196

Why were transposable elements considered to be selfish DNA

No genetic info useful to host

197

What types of TE have poly a tails

LINE and SINE

198

What is an IR

Inverted repeat

199

Where does transposase cut

Between IR and genomic DNA

200

Autonomous TE

Nondeleted TE that can transpose on their own

201

Nonautonomous TE

Can transpose only if transposase activity expressed from intact TE

202

How can TE disrupt genes and alter genomes

-alter phenotype by inserting within gene, coding region
-spontaneous chromosomal arrangements due to unequal crossing over
-gene relocation due to transposition (composite TE formation)

203

Gene relocation due to transposition

Occurs when 2 copies of TE are in nearby locations on same chromosome

204

Why is sex chromosome aneuploidy tolerated

Inactivation of X chromosome

205

Mitotic nondisjunction

sisters dont separate

206

Why are all polyploids with odd number of chromosomes sterile

Cannot produce balanced gametes

207

How are bacteria crucial to environment

-Release oxygen
-Recycle
-Digest waste
-Neutralize pesticides and pollutants
-produce vitamins for organisms

208

Insertion sequences

Like eukaryotic transposable elements
-inverted repeats at ends
-encodes transposase
-can disrupt gene function
-can rearrange bacterial genome (deletions and inversions)

209

E coli characteristics

-inhabits intestines
-can grow w/o oxygen

210

What happens because bacteria are monoploid

All mutations are expressed in their phenotype

211

Another name for lytic phages

Virulent