Phenotypic Mutation 'Wharf' (pdf version)
AlleleWharf
Mutation Type missense
Chromosome11
Coordinate34,623,198 bp (GRCm39)
Base Change T ⇒ G (forward strand)
Gene Dock2
Gene Name dedicator of cyto-kinesis 2
Synonym(s) CED-5, Hch, MBC
Chromosomal Location 34,176,815-34,674,719 bp (-) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The protein encoded by this gene belongs to the CDM protein family. It is specifically expressed in hematopoietic cells and is predominantly expressed in peripheral blood leukocytes. The protein is involved in remodeling of the actin cytoskeleton required for lymphocyte migration in response to chemokine signaling. It activates members of the Rho family of GTPases, for example RAC1 and RAC2, by acting as a guanine nucleotide exchange factor (GEF) to exchange bound GDP for free GTP. [provided by RefSeq, Oct 2016]
PHENOTYPE: Homozygous mutants are defective in the migration of T and B lympohcytes in response to chemokines, and thus display immune defects such as lymphocytopenia, atrophy of lymphoid follicles and loss of marginal-zone B cells. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_033374; MGI:2149010

MappedYes 
Amino Acid Change Histidine changed to Proline
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000090884] [ENSMUSP00000098916] [ENSMUSP00000116893]
AlphaFold Q8C3J5
SMART Domains Protein: ENSMUSP00000090884
Gene: ENSMUSG00000020143
AA Change: H65P

DomainStartEndE-ValueType
SH3 11 68 1.22e-11 SMART
Pfam:DOCK_N 71 414 2e-113 PFAM
Pfam:DOCK-C2 419 616 1e-60 PFAM
Pfam:DHR-2 1114 1614 6.3e-96 PFAM
low complexity region 1691 1706 N/A INTRINSIC
low complexity region 1793 1800 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.263 (Sensitivity: 0.91; Specificity: 0.88)
(Using ENSMUST00000093193)
SMART Domains Protein: ENSMUSP00000098916
Gene: ENSMUSG00000020143
AA Change: H65P

DomainStartEndE-ValueType
SH3 11 68 1.22e-11 SMART
Pfam:DOCK_N 71 414 1.4e-113 PFAM
Pfam:DOCK-C2 419 616 5.5e-61 PFAM
low complexity region 1163 1171 N/A INTRINSIC
Predicted Effect probably benign

PolyPhen 2 Score 0.215 (Sensitivity: 0.92; Specificity: 0.88)
(Using ENSMUST00000101365)
SMART Domains Protein: ENSMUSP00000116893
Gene: ENSMUSG00000020143
AA Change: H65P

DomainStartEndE-ValueType
SH3 11 68 1.22e-11 SMART
Pfam:DOCK-C2 418 617 1.8e-55 PFAM
Predicted Effect possibly damaging

PolyPhen 2 Score 0.813 (Sensitivity: 0.84; Specificity: 0.93)
(Using ENSMUST00000143540)
Meta Mutation Damage Score 0.6766 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(29) : Chemically induced (ENU)(10) Chemically induced (other)(1) Gene trapped(11) Spontaneous(1) Targeted(6)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00334:Dock2 APN 11 34595488 missense probably damaging 1.00
IGL00469:Dock2 APN 11 34179603 splice site probably benign
IGL01061:Dock2 APN 11 34596653 missense probably damaging 1.00
IGL01319:Dock2 APN 11 34589617 missense possibly damaging 0.61
IGL01451:Dock2 APN 11 34260390 missense probably damaging 1.00
IGL01490:Dock2 APN 11 34596608 missense probably damaging 0.97
IGL01601:Dock2 APN 11 34189528 critical splice donor site probably null
IGL01800:Dock2 APN 11 34647100 missense probably damaging 1.00
IGL01804:Dock2 APN 11 34212433 missense probably benign 0.01
IGL01823:Dock2 APN 11 34212391 missense probably damaging 1.00
IGL01829:Dock2 APN 11 34596668 missense probably damaging 0.98
IGL01830:Dock2 APN 11 34582744 nonsense probably null
IGL01835:Dock2 APN 11 34260435 missense possibly damaging 0.51
IGL01845:Dock2 APN 11 34599692 missense probably benign 0.02
IGL01953:Dock2 APN 11 34623183 missense probably benign 0.28
IGL01989:Dock2 APN 11 34218053 missense probably benign
IGL02081:Dock2 APN 11 34204355 missense probably benign
IGL02105:Dock2 APN 11 34605352 missense probably damaging 1.00
IGL02153:Dock2 APN 11 34180670 missense probably benign 0.01
IGL02170:Dock2 APN 11 34217949 missense probably damaging 1.00
IGL02344:Dock2 APN 11 34622337 missense probably damaging 0.98
IGL02389:Dock2 APN 11 34589567 splice site probably benign
IGL02409:Dock2 APN 11 34451204 missense probably benign 0.00
IGL02472:Dock2 APN 11 34199801 missense probably benign 0.00
IGL02625:Dock2 APN 11 34451168 critical splice donor site probably null
IGL02929:Dock2 APN 11 34218048 missense probably damaging 1.00
IGL02951:Dock2 APN 11 34260448 unclassified probably benign
IGL02999:Dock2 APN 11 34583086 missense probably damaging 0.99
IGL03165:Dock2 APN 11 34578360 missense probably damaging 0.99
Arches UTSW 11 34580587 missense probably damaging 1.00
capitol_reef UTSW 11 34244170 critical splice acceptor site probably null
Croesus UTSW 11 34611854 missense probably damaging 1.00
denali UTSW 11 34179472 critical splice donor site probably null
dew UTSW 11 34198636 nonsense probably null
Dinghy UTSW 11 34212460 missense possibly damaging 0.70
Dry UTSW 11 34181652 missense possibly damaging 0.79
frazz UTSW 11 34198572 critical splice donor site probably benign
frizz UTSW 11 34208184 splice site probably benign
gildenstern UTSW 11 34623166 critical splice donor site probably null
godsgrace UTSW 11 34586280 missense probably damaging 1.00
Harborside UTSW 11 34212445 missense probably benign
Landing UTSW 11 34605328 missense possibly damaging 0.83
latest UTSW 11 34647049 missense probably damaging 1.00
Launch UTSW 11 34206562 missense probably damaging 1.00
liaoning UTSW 11 34599620 missense probably damaging 1.00
lucre UTSW 11 34595436 frame shift probably null
midas UTSW 11 34244323 missense probably damaging 0.99
muelle UTSW 11 34578365 missense probably damaging 1.00
narrowest UTSW 11 34232652 missense probably damaging 0.98
pier UTSW 11 34580593 missense probably damaging 1.00
Plank UTSW 11 34674622 missense possibly damaging 0.51
resplendent UTSW 11 34618287 nonsense probably null
riches UTSW 11 34579279 critical splice donor site probably null
skiff UTSW 11 34212388 missense probably null 0.80
Slip UTSW 11 34244286 missense probably benign 0.25
toothskin UTSW 11 34414922 missense probably damaging 1.00
Touch UTSW 11 34223750 missense possibly damaging 0.95
wassup UTSW 11 34453413 missense probably damaging 1.00
BB009:Dock2 UTSW 11 34217998 missense probably benign 0.00
BB019:Dock2 UTSW 11 34217998 missense probably benign 0.00
IGL03052:Dock2 UTSW 11 34182853 missense probably benign 0.01
PIT4377001:Dock2 UTSW 11 34611835 missense probably benign 0.02
R0006:Dock2 UTSW 11 34262453 unclassified probably benign
R0012:Dock2 UTSW 11 34674622 missense possibly damaging 0.51
R0063:Dock2 UTSW 11 34647111 critical splice acceptor site probably null
R0063:Dock2 UTSW 11 34647111 critical splice acceptor site probably null
R0116:Dock2 UTSW 11 34579392 intron probably benign
R0149:Dock2 UTSW 11 34388327 missense probably damaging 1.00
R0361:Dock2 UTSW 11 34388327 missense probably damaging 1.00
R0462:Dock2 UTSW 11 34218052 missense possibly damaging 0.74
R0471:Dock2 UTSW 11 34579380 missense probably benign 0.30
R0538:Dock2 UTSW 11 34595545 splice site probably benign
R0543:Dock2 UTSW 11 34244325 missense probably damaging 1.00
R0660:Dock2 UTSW 11 34198621 missense probably damaging 1.00
R0676:Dock2 UTSW 11 34586063 missense probably damaging 0.99
R0722:Dock2 UTSW 11 34414970 splice site probably benign
R0801:Dock2 UTSW 11 34599620 missense probably damaging 1.00
R1110:Dock2 UTSW 11 34206535 missense possibly damaging 0.78
R1171:Dock2 UTSW 11 34586068 missense probably damaging 1.00
R1387:Dock2 UTSW 11 34223309 splice site probably benign
R1445:Dock2 UTSW 11 34189705 missense probably benign
R1494:Dock2 UTSW 11 34232761 nonsense probably null
R1589:Dock2 UTSW 11 34597288 missense probably damaging 0.99
R1597:Dock2 UTSW 11 34595474 missense probably benign 0.00
R1629:Dock2 UTSW 11 34212480 splice site probably null
R1749:Dock2 UTSW 11 34182767 critical splice donor site probably null
R1888:Dock2 UTSW 11 34598169 missense probably damaging 1.00
R1888:Dock2 UTSW 11 34598169 missense probably damaging 1.00
R1899:Dock2 UTSW 11 34244286 missense probably benign 0.25
R1924:Dock2 UTSW 11 34414934 missense possibly damaging 0.69
R2031:Dock2 UTSW 11 34618297 splice site probably benign
R2045:Dock2 UTSW 11 34244106 splice site probably null
R2098:Dock2 UTSW 11 34609832 missense probably damaging 0.99
R2098:Dock2 UTSW 11 34216279 missense probably benign 0.16
R2129:Dock2 UTSW 11 34618242 missense probably damaging 1.00
R2147:Dock2 UTSW 11 34179472 critical splice donor site probably null
R2149:Dock2 UTSW 11 34179472 critical splice donor site probably null
R2150:Dock2 UTSW 11 34179472 critical splice donor site probably null
R2176:Dock2 UTSW 11 34586044 missense probably benign 0.00
R2230:Dock2 UTSW 11 34244323 missense probably damaging 0.99
R2508:Dock2 UTSW 11 34262485 missense probably benign 0.04
R2875:Dock2 UTSW 11 34609712 missense probably damaging 1.00
R2885:Dock2 UTSW 11 34580593 missense probably damaging 1.00
R2910:Dock2 UTSW 11 34182910 splice site probably benign
R3081:Dock2 UTSW 11 34181610 missense probably benign
R3418:Dock2 UTSW 11 34580587 missense probably damaging 1.00
R3552:Dock2 UTSW 11 34611787 missense probably benign 0.22
R3731:Dock2 UTSW 11 34599722 missense probably damaging 1.00
R3846:Dock2 UTSW 11 34623198 missense possibly damaging 0.81
R4135:Dock2 UTSW 11 34605328 missense possibly damaging 0.83
R4598:Dock2 UTSW 11 34189536 missense probably damaging 1.00
R4599:Dock2 UTSW 11 34189536 missense probably damaging 1.00
R4715:Dock2 UTSW 11 34244118 missense probably damaging 1.00
R4722:Dock2 UTSW 11 34586298 missense probably damaging 1.00
R4742:Dock2 UTSW 11 34244170 critical splice acceptor site probably null
R4830:Dock2 UTSW 11 34223767 splice site probably null
R4884:Dock2 UTSW 11 34216248 missense probably damaging 1.00
R4990:Dock2 UTSW 11 34586078 missense probably damaging 1.00
R5334:Dock2 UTSW 11 34178643 missense probably benign 0.00
R5570:Dock2 UTSW 11 34618233 missense probably damaging 1.00
R5602:Dock2 UTSW 11 34204391 missense probably benign 0.16
R5681:Dock2 UTSW 11 34199836 missense probably benign 0.06
R5809:Dock2 UTSW 11 34212445 missense probably benign
R5860:Dock2 UTSW 11 34206562 missense probably damaging 1.00
R6111:Dock2 UTSW 11 34599614 missense probably damaging 0.99
R6155:Dock2 UTSW 11 34244123 missense probably benign 0.06
R6156:Dock2 UTSW 11 34197789 missense possibly damaging 0.51
R6173:Dock2 UTSW 11 34212388 missense probably null 0.80
R6182:Dock2 UTSW 11 34179476 missense probably damaging 0.97
R6188:Dock2 UTSW 11 34453396 missense probably damaging 0.98
R6191:Dock2 UTSW 11 34181652 missense possibly damaging 0.79
R6283:Dock2 UTSW 11 34598152 missense probably damaging 0.99
R6395:Dock2 UTSW 11 34182874 missense probably damaging 1.00
R6465:Dock2 UTSW 11 34453413 missense probably damaging 1.00
R6500:Dock2 UTSW 11 34312822 missense possibly damaging 0.76
R6561:Dock2 UTSW 11 34578365 missense probably damaging 1.00
R6745:Dock2 UTSW 11 34596670 missense probably damaging 1.00
R6745:Dock2 UTSW 11 34596669 missense probably damaging 1.00
R6880:Dock2 UTSW 11 34579279 critical splice donor site probably null
R6913:Dock2 UTSW 11 34647049 missense probably damaging 1.00
R6997:Dock2 UTSW 11 34414922 missense probably damaging 1.00
R7057:Dock2 UTSW 11 34586044 missense probably benign 0.00
R7057:Dock2 UTSW 11 34177684 missense probably benign 0.10
R7134:Dock2 UTSW 11 34260363 missense probably benign 0.03
R7188:Dock2 UTSW 11 34189675 missense possibly damaging 0.87
R7239:Dock2 UTSW 11 34181677 missense probably benign 0.00
R7247:Dock2 UTSW 11 34605340 nonsense probably null
R7250:Dock2 UTSW 11 34586120 missense probably damaging 1.00
R7250:Dock2 UTSW 11 34586032 missense probably benign 0.01
R7271:Dock2 UTSW 11 34223750 missense possibly damaging 0.95
R7284:Dock2 UTSW 11 34180672 missense probably benign 0.01
R7397:Dock2 UTSW 11 34609816 missense probably benign 0.00
R7464:Dock2 UTSW 11 34586105 missense probably damaging 0.99
R7512:Dock2 UTSW 11 34262542 missense possibly damaging 0.95
R7556:Dock2 UTSW 11 34611778 missense probably benign 0.43
R7663:Dock2 UTSW 11 34611854 missense probably damaging 1.00
R7779:Dock2 UTSW 11 34605282 missense probably benign 0.38
R7797:Dock2 UTSW 11 34232652 missense probably damaging 0.98
R7855:Dock2 UTSW 11 34223698 missense probably damaging 1.00
R7922:Dock2 UTSW 11 34598154 missense probably benign 0.29
R7932:Dock2 UTSW 11 34217998 missense probably benign 0.00
R8013:Dock2 UTSW 11 34596677 missense probably damaging 0.96
R8192:Dock2 UTSW 11 34623166 critical splice donor site probably null
R8244:Dock2 UTSW 11 34586280 missense probably damaging 1.00
R8307:Dock2 UTSW 11 34260362 missense possibly damaging 0.95
R8418:Dock2 UTSW 11 34609795 missense probably benign 0.01
R8460:Dock2 UTSW 11 34180825 critical splice acceptor site probably null
R8495:Dock2 UTSW 11 34181622 missense probably benign 0.14
R8556:Dock2 UTSW 11 34212457 missense possibly damaging 0.84
R8690:Dock2 UTSW 11 34618287 nonsense probably null
R8743:Dock2 UTSW 11 34223252 nonsense probably null
R8757:Dock2 UTSW 11 34586067 missense probably benign 0.13
R8759:Dock2 UTSW 11 34586067 missense probably benign 0.13
R8793:Dock2 UTSW 11 34451215 missense probably benign 0.00
R8882:Dock2 UTSW 11 34595436 frame shift probably null
R8885:Dock2 UTSW 11 34260396 missense probably benign 0.01
R8943:Dock2 UTSW 11 34599646 missense possibly damaging 0.63
R9171:Dock2 UTSW 11 34589670 missense probably benign 0.12
R9182:Dock2 UTSW 11 34260398 missense possibly damaging 0.51
R9203:Dock2 UTSW 11 34622366 missense possibly damaging 0.92
R9310:Dock2 UTSW 11 34244139 missense possibly damaging 0.71
R9388:Dock2 UTSW 11 34212460 missense possibly damaging 0.70
R9490:Dock2 UTSW 11 34589582 missense possibly damaging 0.90
R9568:Dock2 UTSW 11 34599638 missense possibly damaging 0.83
R9593:Dock2 UTSW 11 34178607 missense probably benign 0.34
R9694:Dock2 UTSW 11 34218054 missense probably benign
R9697:Dock2 UTSW 11 34204417 missense probably benign
R9753:Dock2 UTSW 11 34223673 missense possibly damaging 0.68
R9783:Dock2 UTSW 11 34208128 missense possibly damaging 0.83
X0017:Dock2 UTSW 11 34216271 missense probably benign 0.08
X0018:Dock2 UTSW 11 34182833 missense possibly damaging 0.65
X0058:Dock2 UTSW 11 34206564 missense probably damaging 1.00
X0066:Dock2 UTSW 11 34260357 missense possibly damaging 0.95
Z1088:Dock2 UTSW 11 34583209 missense probably damaging 1.00
Z1088:Dock2 UTSW 11 34388300 missense probably benign 0.14
Z1088:Dock2 UTSW 11 34586039 nonsense probably null
Z1176:Dock2 UTSW 11 34609751 missense probably benign 0.04
Z1177:Dock2 UTSW 11 34262553 missense possibly damaging 0.68
Mode of Inheritance Unknown
Local Stock
Repository
Last Updated 2019-09-04 9:31 PM by Diantha La Vine
Record Created 2019-01-22 10:59 AM by Bruce Beutler
Record Posted 2019-02-01
Phenotypic Description

Figure 1. Wharf mice exhibit decreased frequencies of peripheral B1b cells. Flow cytometric analysis of peripheral blood was utilized to determine B1b cell frequency. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

Figure 2. Wharf mice exhibit increased frequencies of peripheral NK cells. Flow cytometric analysis of peripheral blood was utilized to determine NK cell frequency. Raw data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The Wharf phenotype was identified among G3 mice of the pedigree R3846, some of which showed reduced frequencies of B1b cells (Figure 1) with concomitant increased frequencies of NK cells (Figure 1) in the peripheral blood.

Nature of Mutation

Figure 3. Linkage mapping of the reduced B1b cell frequency using a dominant model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 38 mutations (X-axis) identified in the G1 male of pedigree R3846. Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 38 mutations. Both of the above anomalies were linked by continuous variable mapping to a mutation in Dock2: an A to C transversion at base pair 34,732,371 (v38) on chromosome 11, or base pair 51,539 in the GenBank genomic region NC_000077 for Dock2. The strongest association was found with an unknown (additive/dominant) model of inheritance to the normalized B1b cell frequency, wherein nine heterozygous mice departed phenotypically from 11 homozygous reference mice with a P value of 0.000424  (Figure 3); no homozygous variant mice were available/alive at time of screening.

The mutation corresponds to residue 276 in the mRNA sequence NM_178666 within exon 4 of 52 total exons.

260 CCTACATCCTTTATCCATCTCAAGGAAGTGACA

60  -P--T--S--F--I--H--L--K--E--V--T-

The mutated nucleotide is indicated in red. The mutation results in a histidine to proline substitution at position 65 (H65P) in the DOCK2 protein, and is strongly predicted by Polyphen-2 to be benign (score = 0.263).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 4. Domain structure of mouse DOCK2. DOCK2 contains an N-terminal SH3 domain. SH3-containing DOCK proteins have been shown to interact physically with the scaffolding proteins engulfment and cell motility protein 1 (ELMO1) and ELMO2, significantly promoting Rac activation. The DHR-1 domain shares weak homology to the C2 domain. The large DHR-2 domain interacts with the nucleotide-free form of Rac. The Wharf mutation is indicated in red. This image is interactive. Other mutations found in DOCK2 are noted in red. Click on each mutation for more specific information.

DOCK2 belongs to the DOCK180 superfamily of guanine nucleotide exchange factors (GEFs) that have been shown to activate members of the Rho family of small GTPases [Figure 4; (1-4)]. Members of the DOCK A and B groups contain an N-terminal SH3 domain. Two domains are shared amongst all DOCK proteins, the catalytic DHR-2 (DOCK homology region 2) or CZH-2 (CDM-zizimin homology 2) domain (amino acids 1114-1620 in DOCK2) and the DHR-1 or CZH-1 domain (amino acids 420-662 in DOCK2) (2;4).  Several DOCK proteins, including DOCK2, appear to be localized to the plasma membrane via interaction of the DHR-1 domain with phosphatidylinositol (3,5)-biphosphate [PtdIns(3,5)P2] and phosphatidylinositol (3,4,5) P3 (PIP3) signaling lipids (5;6). The DHR-2 domains of several DOCK family members interact with the nucleotide-free form of Rac and/or Cdc42 (2;4). DOCK2 has an N-terminal SH3 domain (amino acids 8-69).

The Wharf mutation results in a histidine to proline substitution at position 65 (H65P); His65 is within the SH3 domain.

See the record frazz for more information on Dock2.

Putative Mechanism

Rho GTPases are known regulators of the actin cytoskeleton and affect multiple cellular activities including cell morphology, polarity, migration, proliferation and apoptosis, phagocytosis, cytokinesis, adhesion, vesicular transport, transcription and neurite extension and retraction. Regulation of Rho GTPase activity involves the GEFs that promote the exchange of GDP for GTP, the GTPase-activating proteins (GAPs) that enhance the GTPase activity of Rho proteins, and the Rho guanine nucleotide-dissociation inhibitors (RhoGDIs) that sequester Rho GTPases in a GDP-bound state. DOCK2 functions in the polarization and migration of immune cell subsets.  DOCK2 functions downstream of chemokine receptors to regulate Rac activation and migration of B and T lymphocytes, neutrophils, plasmacytoid dendritic cells (pDCs), and hematopoietic stem cells, but not monocytes or other myeloid cell types (8-10).  The immune cells affected by DOCK2 deficiency display aberrant homing, activation, adhesion, polarization and migration. DOCK2-deficient lymphocytes fail to respond normally to the chemokines CCL21, CCL19, CXCL13, and CXCL12, resulting in impaired homing to secondary lymphoid organs and aberrant morphology of these tissues (8)Dock2 -/ -mice display a number of immunological phenotypes including lack of marginal zone B cells, reduced numbers of mature CD4+ T cells and the major subset of natural killer T (NKT) cells expressing the semi-invariant Vα14 T cell receptor (TCR), and aberrant TCR signaling (8;11;12).  The phenotype of the Wharf mice indicates loss of DOCK2-assocated function.

Primers PCR Primer
Wharf_pcr_F: GGAGGACTTTACAGGACTGTTCC
Wharf_pcr_R: GAATCTGTGCCTGCTCCATC

Sequencing Primer
Wharf_seq_F: CTCAGCTCAGCACTGGGATTATAAG
Wharf_seq_R: CCTGCTCCATCTAATTCTCAGATTAG
Genotyping

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold


The following sequence of 536 nucleotides is amplified (chromosome 11, - strand):


1   gaatctgtgc ctgctccatc taattctcag attagtcttt gaaagttttg tggtttcaga
61  agccagtggc ttagcagggg aaggaaactg gaaaacaagg agtctgttat ttttctatga
121 cttgtcttcc atcctattta ttgtgtttcc tacagggcat tttccctaca tcctttatcc
181 atctcaagga agtgacagtg gagaagagaa ggtacttgtg gggactgggg agggactcag
241 ttggtaaagt gcctgccttg tggggaccag ggagggactc agttggtaaa gtgtctgcct
301 tgtaagcatg acccaagctg agttccttca gaacacatgt gaaaggagct gagtgaaatg
361 ctatatactt ataatcccag tgctgagctg agggctggga tgacagccgg gtgcctgagg
421 cccttcctgc ttgtcaaggt ccaggctagc gcaagagtcc gtctcaaaac aaaactaaag
481 ccacaaacag caaagggagg gggttgcccc tgaggaacag tcctgtaaag tcctcc


Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References
Science Writers Anne Murray
Illustrators Diantha La Vine
AuthorsJin Huk Choi, Xue Zhong, and Bruce Beutler